Immune Assisted Tissue Engineering via Incorporation of Macrophages in Cell-Laden Hydrogels Under Cytokine Stimulation

The function of soft tissues is intricately linked to their connections with the other systems of the body such as circulation, nervous system, and immune system. The presence of resident macrophages in tissues provides a means to control tissue homeostasis and also a way to react to the physical/biological insults and tissue damage. Thus, incorporation of resident macrophage like phenotype-controlled macrophages in engineered tissues can improve their fidelity as model tissues and also improve their rate of integration and facilitate the resolution of inflammation for regenerative medicine applications. Herein, we demonstrate two potential ways to immunoassist the remodeling process of engineered soft tissues in three-dimensional (3-D) gelatin based hydrogels containing fibroblasts and/or endothelial cells: (i) with supplementation of interleukin-4 (IL-4) in the presence of macrophages and (ii) in tri-culture via naive monocytes or differentiated macrophages. The presence of IL-4 had a proliferative effect on fibroblasts, with a significant boosting effect on proliferation and cytokine secretion in the presence of differentiated macrophages with an upregulation of activin, interleukin-1 receptor antagonist (IL-1RA), tumor necrosis factor alpha (TNF-α), and interleukin-1 beta (IL-1β), creating a more stimulating microenvironment. The addition of IL-4 in endothelial cell/macrophage co-culture configuration improved the organization of the sprout-like structures, with a boost in proliferation at day 1 and with an upregulation of IL-6 and IL-1RA at the earliest stage in the presence of differentiated macrophages creating a favorable microenvironment for angiogenesis. In tri-culture conditions, the presence of monocytes or macrophages resulted in a denser tissue-like structure with highly remodeled hydrogels. The presence of differentiated macrophages had a boosting effect on the angiogenic secretory microenvironment, such as IL-6 and IL-8, without any additional cytokine supplementation. The presence of fibroblasts in combination with endothelial cells also had a significant effect on the secretion of angiopoietin. Our results demonstrate that incorporation of macrophages in a resident macrophage function and their phenotype control have significant effects on the maturation and cytokine microenvironment of 3-D multiple cell type-laden hydrogels, which can be harnessed for better integration of implantable systems and for more physiologically relevant in vitro tissue models with an immune component

Investigation of the mechanical and chemical characteristics of nanotubular and nano-pitted anodic films on grade 2 titanium dental implant materials

Abstract Objective The objective of this study was to investigate the reproducibility, mechanical integrity, surface characteristics and corrosion behavior of nanotubular (NT) titanium oxide arrays in comparison with a novel nano-pitted (NP) anodic film. Methods Surface treatment processes were developed to grow homogenous NT and NP anodic films on the surface of grade 2 titanium discs and dental implants. The effect of process parameters on the surface characteristics and reproducibility of the anodic films was investigated and optimized. The mechanical integrity of the NT and NP anodic films were investigated by scanning electron microscopy, surface roughness measurement, scratch resistance and screwing tests, while the chemical and physicochemical properties were investigated in corrosion tests, contact angle measurement and X-ray photoelectron spectroscopy (XPS). Results and discussion The growth of NT anodic films was highly affected by process parameters, especially by temperature, and they were apt to corrosion and exfoliation. In contrast, the anodic growth of NP film showed high reproducibility even on the surface of 3-dimensional screw dental implants and they did not show signs of corrosion and exfoliation. The underlying reason of the difference in the tendency for exfoliation of the NT and NP anodic films is unclear; however the XPS analysis revealed fluorine dopants in a magnitude larger concentration on NT anodic film than on NP surface, which was identified as a possible causative. Concerning other surface characteristics that are supposed to affect the biological behavior of titanium implants, surface roughness values were found to be similar, whereas considerable differences were revealed in the wettability of the NT and NP anodic films. Conclusion Our findings suggest that the applicability of NT anodic films on the surface of titanium bone implants may be limited because of mechanical considerations. In contrast, it is worth to consider the applicability of nano-pitted anodic films over nanotubular arrays for the enhancement of the biological properties of titanium implants

The impact of surface chemistry modification on macrophage polarisation

Macrophages are innate immune cells that have a central role in combating infection and maintaining tissue homeostasis. They exhibit remarkable plasticity in response to environmental cues. At either end of a broad activation spectrum are pro-inflammatory (M1) and anti-inflammatory (M2) macrophages with distinct functional and phenotypical characteristics. Macrophages also play a crucial role in orchestrating immune responses to biomaterials used in the fabrication of implantable devices and drug delivery systems. To assess the impact of different surface chemistries on macrophage polarisation, human monocytes were cultured for 6 days on untreated hydrophobic polystyrene (PS) and hydrophilic O2 plasma-etched polystyrene (O2-PS40) surface. Our data clearly show that monocytes cultured on the hydrophilic O2-PS40 surface are polarised towards an M1-like phenotype, as evidenced by significantly higher expression of the pro-inflammatory transcription factors STAT1 and IRF5. By comparison, monocytes cultured on the hydrophobic PS surface exhibited an M2-like phenotype with high expression of mannose receptor (MR) and production of the anti-inflammatory cytokines IL-10 and CCL18. While the molecular basis of such different patterns of cell differentiation is yet to be fully elucidated, we hypothesise that it is due to the adsorption of different biomolecules on these surface chemistries. Indeed our surface characterisation data show quantitative and qualitative differences between the protein layers on that the O2-PS40 surface compared to PS surface which could be responsible for the observed differential macrophage polarisation on each surface

Dual growth factor delivery using PLGA nanoparticles in silk fibroin/PEGDMA hydrogels for articular cartilage tissue engineering

Degeneration of articular cartilage due to damages, diseases, or age-related factors can significantly decrease the mobility of the patients. Various tissue engineering approaches which take advantage of stem cells and growth factors in a three-dimensional constructs have been used for reconstructing articular tissue. Proliferative impact of basic fibroblast growth factor (bFGF) and chondrogenic differentiation effect of transforming growth factor-beta 1 (TGF-beta 1) over mesenchymal stem cells have previously been verified. In this study, silk fibroin (SF) and of poly(ethylene glycol) dimethacrylate (PEGDMA) were used to provide a versatile platform for preparing hydrogels with tunable mechanical, swelling and degradation properties through physical and chemical crosslinking as a microenvironment for chondrogenic differentiation in the presence of bFGF and TGF-beta 1 releasing nanoparticles (NPs) for the first time. Scaffolds with compressive moduli ranging from 95.70 +/- 17.82 to 338.05 +/- 38.24 kPa were obtained by changing both concentration PEGDMA and volume ratio of PEGDMA with 8% SF. Highest cell viability was observed in PEGDMA 10%-SF 8% (1:1) [PEG10-SF8(1:1)] hydrogel group. Release of bFGF and TGF-beta 1 within PEG10-SF8(1:1) hydrogels resulted in higher DNA and glycosaminoglycans amounts indicating synergistic effect of dual release over proliferation and chondrogenic differentiation of dental pulp stem cells in hydrogels, respectively. Our results suggested that simultaneous delivery of bFGF and TGF-beta 1 through utilization of PLGA NPs within PEG10-SF8(1:1) hydrogel provided a novel and versatile means for articular cartilage regeneration as they allow for dosage- and site-specific multiple growth factor delivery

Effect of human corneal keratocytes and retinal pigment epithelial cells on the mechanical properties of micropatterned collagen films

Collagen-based micropatterned films were seeded with human corneal keratocyte and epithelial cells to study their mechanical properties as tissue engineering substrates. The patterns were in the form of parallel channels with slanted walls. Influence of cell presence, type and growth on the mechanical properties of the films was investigated. Unseeded films showed gradual strength reduction from an initial value of 0.046 N/mm, possibly due to degradation, down to 0.032 +/- 0.012 N/mm in 2 weeks. Keratocyte growth was found to significantly improve the mechanical behavior of the films upon 1 week of incubation (0.067 +/- 0.017N/mm) and the improvement continued gradually over the next 2 weeks. Films seeded with D407 retinal pigment epithelial cells, on the other hand, experienced a decrease (0.023 +/- 0.011 N/mm), followed by a slight increase in mechanical properties in the 21-day period. A steady increase in the number of keratocytes along the channels, cytoskeleton alignment and extracellular matrix (ECM) secretion restricted to the channels was observed. Increase in strength observed with keratocytes and, to a lesser extent, with the epithelial cells can be attributed to directional ECM synthesis and the orientation of the cells and their cytoskeleton which contribute to the strength in the direction of the channels. This study showed that cell, especially keratocyte, presence compensates for the degradation of collagen films and improve the overall mechanical properties of the engineered tissue

Engineering functional epithelium for regenerative medicine and in vitro organ models: A review

Recent advances in the fields of microfabrication, biomaterials, and tissue engineering have provided new opportunities for developing biomimetic and functional tissues with potential applications in disease modeling, drug discovery, and replacing damaged tissues. An intact epithelium plays an indispensable role in the functionality of several organs such as the trachea, esophagus, and cornea. Furthermore, the integrity of the epithelial barrier and its degree of differentiation would define the level of success in tissue engineering of other organs such as the bladder and the skin. In this review, we focus on the challenges and requirements associated with engineering of epithelial layers in different tissues. Functional epithelial layers can be achieved by methods such as cell sheets, cell homing, and in situ epithelialization. However, for organs composed of several tissues, other important factors such as (1) in vivo epithelial cell migration, (2) multicell-type differentiation within the epithelium, and (3) epithelial cell interactions with the underlying mesenchymal cells should also be considered. Recent successful clinical trials in tissue engineering of the trachea have highlighted the importance of a functional epithelium for long-term success and survival of tissue replacements. Hence, using the trachea as a model tissue in clinical use, we describe the optimal structure of an artificial epithelium as well as challenges of obtaining a fully functional epithelium in macroscale. One of the possible remedies to address such challenges is the use of bottom-up fabrication methods to obtain a functional epithelium. Modular approaches for the generation of functional epithelial layers are reviewed and other emerging applications of microscale epithelial tissue models for studying epithelial/mesenchymal interactions in healthy and diseased (e.g., cancer) tissues are described. These models can elucidate the epithelial/mesenchymal tissue interactions at the microscale and provide the necessary tools for the next generation of multicellular engineered tissues and organ-on-a-chip systems. © Copyright 2013, Mary Ann Liebert, Inc. 2013

Mitigating the foreign body response through ‘immune-instructive’ biomaterials

ObjectivesBiomaterials are routinely used in clinical applications. A key to the clinical success of implanted biomaterials is not eliciting detrimental immune responses. In this article, we provide an overview of immune responses to biomaterials, along with biomaterial-based approaches to mitigate the adverse host reactions while supporting pro-healing immune responses. We also review existing in-vitro models used to assess the biocompatibility of biomaterials.Key findingsOnce implanted, biomaterials are often detected as foreign bodies by the immune system, triggering detrimental immune responses. Such responses could damage host tissues and impair the function of implanted materials or devices. Therefore, there is substantial interest in developing new materials and tools with the ability to modulate immune responses to support tissue regeneration and healing processes. However, the bioengineering of immune responses through biomaterials requires detailed understanding of how the immune system typically responds to foreign materials. This knowledge can inform designing materials with bio-instructive chemistries and/or surface attributes. In this review, first we briefly discuss basic aspects of the foreign body response followed by different strategies for developing ‘immune-instructive’ biomaterials, models to test their efficacy and examples of their clinical applications.ConclusionsPromising progress has been made in the field of biomaterial engineering however, how different immune cells interact with biomaterials is yet to be fully elucidated. A better understanding of cell-material interactions, and particularly the impact of inter-individual variations, will allow the development of new generation of more personalised ‘immune-instructive’ biomaterials and medical devices to modulate immune responses towards anti-inflammatory and pro-healing phenotypes

Polyarginine Decorated Polydopamine Nanoparticles With Antimicrobial Properties for Functionalization of Hydrogels

Polydopamine (PDA) nanoparticles are versatile structures that can be stabilized with proteins. In this study, we have demonstrated the feasibility of developing PDA/polypeptides complexes in the shape of nanoparticles. The polypeptide can also render the nanoparticle functional. Herein, we have developed antimicrobial nanoparticles with a narrow size distribution by decorating the polydopamine particles with a chain-length controlled antimicrobial agent Polyarginine (PAR). The obtained particles were 3.9 ± 1.7 nm in diameter and were not cytotoxic at 1:20 dilution and above. PAR-decorated nanoparticles have exhibited a strong antimicrobial activity against S. aureus, one of the most common pathogen involved in implant infections. The minimum inhibitory concentration is 5 times less than the cytotoxicity levels. Then, PAR-decorated nanoparticles have been incorporated into gelatin hydrogels used as a model of tissue engineering scaffolds. These nanoparticles have given hydrogels strong antimicrobial properties without affecting their stability and biocompatibility while improving their mechanical properties (modulus of increased storage). Decorated polydopamine nanoparticles can be a versatile tool for the functionalization of hydrogels in regenerative medicine applications by providing bioactive properties