Plasma polymerised nanoscale coatings of controlled thickness for efficient solid-phase presentation of growth factors

The engineering of biomaterial surfaces and scaffolds for specific biomedical and clinical application is of growing interest. Certain functionalised surfaces can capture and deliver bioactive molecules, such as growth factors (GF), enhancing the clinical efficacy of such systems. With a custom-made plasma polymerisation reactor described here we have developed bioactive polymer coatings based on poly(ethyl acrylate) (PEA). This remarkable polymer unfolds fibronectin (FN) upon adsorption to allow the GF binding region of FN to sequester and present GFs with high efficiency. We systematically evaluate process conditions and their impact on plasma polymerised PEA coatings and characterise the effect of plasma power and deposition time on thickness, wettability and chemical composition of the coatings. We demonstrate that functional substrate roughness can be maintained after deposition of the polymer coatings. Importantly, we show that coatings deposited at different conditions all maintain a similar or better bioactivity than spin coated PEA references. We show that in PEA plasma polymerised coatings FN assembles into nanonetworks with high availability of integrin and GF binding regions that sequester bone morphogenetic protein-2 (BMP-2). We also report similar mesenchymal stem cell adhesion behaviour, as characterised by focal adhesions, and differentiation potential on BMP-2 coated surfaces, regardless of plasma deposition conditions. This is a potent and versatile technology that can help facilitate the use of GFs in clinical applications

Keeping it organ-ized: multicompartment constructs to mimic tissue heterogeneity

Tissue engineering aims at replicating tissues and organs to develop applications in vivo and in vitro. In vivo, by engineering artificial constructs using functional materials and cells to provide both physiological form and function. In vitro, by engineering 3D models to support drug discovery and enable understanding of fundamental biology. 3D culture constructs mimic cell-cell and cell-matrix interactions and use biomaterials seeking to increase the resemblance of engineered tissues with its in vivo homologues. Native tissues, however, include complex architectures, with compartmentalized regions of different properties containing different types of cells that can be captured by multicompartment constructs. Recent advances in fabrication technologies, such as micropatterning, microfluidics or 3D bioprinting, have enabled compartmentalized structures with defined compositions and properties that are essential in creating 3D cell-laden multiphasic complex architectures. This review focuses on advances in engineered multicompartment constructs that mimic tissue heterogeneity. It included multiphasic 3D implantable scaffolds and in vitro models, including systems that incorporate different regions emulating in vivo tissues, highlighting the emergence and relevance of 3D bioprinting in the future of biological research and medicine

Improving cartilage phenotype from differentiated pericytes in tunable peptide hydrogels

Differentiation of stem cells to chondrocytes in vitro usually results in a heterogeneous phenotype. This is evident in the often detected over expression of type X collagen which, in hyaline cartilage structure is not characteristic of the mid-zone but of the deep-zone ossifying tissue. Methods to better match cartilage developed in vitro to characteristic in vivo features are therefore highly desirable in regenerative medicine. This study compares phenotype characteristics between pericytes, obtained from human adipose tissue, differentiated using diphenylalanine/serine (F2/S) peptide hydrogels with the more widely used chemical induced method for chondrogenesis. Significantly higher levels of type II collagen were noted when pericytes undergo chondrogenesis in the hydrogel in the absence of induction media. There is also a balanced expression of collagen relative to aggrecan production, a feature which was biased toward collagen production when cells were cultured with induction media. Lastly, metabolic profiles of each system show considerable overlap between both differentiation methods but subtle differences which potentially give rise to their resultant phenotype can be ascertained. The study highlights how material and chemical alterations in the cellular microenvironment have wide ranging effects on resultant tissue type

Nanotopography influences host–pathogen quorum sensing and facilitates selection of bioactive metabolites in mesenchymal stromal cells and pseudomonas aeruginosa co-cultures

Orthopedic implant-related bacterial infections and resultant antibiotic-resistant biofilms hinder implant-tissue integration and failure. Biofilm quorum sensing (QS) communication determines the pathogen colonization success. However, it remains unclear how implant modifications and host cells are influenced by, or influence, QS. High aspect ratio nanotopographies have shown to reduce biofilm formation of Pseudomonas aeruginosa, a sepsis causing pathogen with well-defined QS molecules. Producing such nanotopographies in relevant orthopedic materials (i.e., titanium) allows for probing QS using mass spectrometry-based metabolomics. However, nanotopographies can reduce host cell adhesion and regeneration. Therefore, we developed a polymer (poly(ethyl acrylate), PEA) coating that organizes extracellular matrix proteins, promoting bioactivity to host cells such as human mesenchymal stromal cells (hMSCs), maintaining biofilm reduction. This allowed us to investigate how hMSCs, after winning the race for the surface against pathogenic cells, interact with the biofilm. Our approach revealed that nanotopographies reduced major virulence pathways, such as LasR. The enhanced hMSCs support provided by the coated nanotopographies was shown to suppress virulence pathways and biofilm formation. Finally, we selected bioactive metabolites and demonstrated that these could be used as adjuncts to the nanostructured surfaces to reduce biofilm formation and enhance hMSC activity. These surfaces make excellent models to study hMSC-pathogen interactions and could be envisaged for use in novel orthopedic implants.</p

Considerations of growth factor and material use in bone tissue engineering using biodegradable scaffolds in vitro and in vivo

Bone tissue engineering aims to harness materials to develop functional bone tissue to heal ‘critical-sized’ bone defects. This study examined a robust, coated poly(caprolactone) trimethacrylate (PCL-TMA) 3D-printable scaffold designed to augment bone formation. Following optimisation of the coatings, three bioactive coatings were examined, i) elastin-like polypeptide (ELP), ii) poly(ethyl acrylate) (PEA), fibronectin (FN) and bone morphogenetic protein-2 (BMP-2) applied sequentially (PEA/FN/BMP-2) and iii) both ELP and PEA/FN/BMP-2 coatings applied concurrently. The scaffold material was robust and showed biodegradability. The coatings demonstrated a significant (p < 0.05) osteogenic response in vitro in alkaline phosphatase gene upregulation and alkaline phosphatase production. The PCL-TMA scaffold and coatings supported angiogenesis and displayed excellent biocompatibility following evaluation on the chorioallantoic membrane assay. No significant (p < 0.05) heterotopic bone formed on the scaffolds within a murine subcutaneous implantation model, compared to the positive control of BMP-2 loaded collagen sponge following examination by micro-computed tomography or histology. The current studies demonstrate a range of innovative coated scaffold constructs with in vitro efficacy and clearly illustrate the importance of an appropriate in vivo environment to validate in vitro functionality prior to scale up and preclinical application

Hybrid micro-/nanoprotein platform provides endocrine-like and extracellular matrix-like cell delivery of growth factors

Protein materials are versatile tools in diverse biomedical fields. Among them, artificial secretory granules (SGs), mimicking those from the endocrine system, act as mechanically stable reservoirs for the sustained release of proteins as oligomeric functional nanoparticles. Only validated in oncology, the physicochemical properties of SGs, along with their combined drug-releasing and scaffolding abilities, make them suitable as smart topographies in regenerative medicine for the prolonged delivery of growth factors (GFs). Thus, considering the need for novel, safe, and cost-effective materials to present GFs, in this study, we aimed to biofabricate a protein platform combining both endocrine-like and extracellular matrix fibronectin-derived (ECM-FN) systems. This approach is based on the sustained delivery of a nanostructured histidine-tagged version of human fibroblast growth factor 2. The GF is presented onto polymeric surfaces, interacting with FN to spontaneously generate nanonetworks that absorb and present the GF in the solid state, to modulate mesenchymal stromal cell (MSC) behavior. The results show that SGs-based topographies trigger high rates of MSCs proliferation while preventing differentiation. While this could be useful in cell therapy manufacture demanding large numbers of unspecialized MSCs, it fully validates the hybrid platform as a convenient setup for the design of biologically active hybrid surfaces and in tissue engineering for the controlled manipulation of mammalian cell growth

Tissue engineered scaffolds for mimetic autografts

Introduction: Despite its regenerative capacity, bone healing can be compromised, leading to delayed fracture regeneration and nonunion. Due to the scarcity of bone tissue that can be used as autograft, novel tissue engineering strategies arise as a promising solution by using biocompatible materials. Methods: Our objective is the development of engineered autografts capable of efficiently treat fracture nonunion. For this purpose, we designed polycaprolactone (PCL) autografts surrounded by a porous membrane mimicking periosteum. To assess their regenerative capacity, these scaffolds were tested in critical size femur defect for ten weeks carrying out μCT and histological analysis. Additionally, we are focusing on the generation of PCL biocomposites, such as poly ethyl-acrylate (PEA) covered PCL membranes which can enhance morphogen functionalization, reducing the effective BMP dose. Results: At the mCT level, structural mimetic PCL scaffolds, showed no significant difference in bone healing (Empty group, 11.47±4.93 mm3; MA, 14.95±3.09 mm3, p=0.1711). Histological analysis demonstrates that MEW PCL mimicking periosteum enhances bone growth, but insufficient for successful healing. However, once functionalized with PEA and BMP-2, these implants showed highly improved regeneration (CTL group, 11,47±4,93 mm3; BMP-2 group, 49,24±13,20 mm3, p = 0.0001). Figure 1. These implants were loaded with BMP-2 solutions previously studied in vitro to estimate morphogen dose, which resulted in 55.64±14.83 ng (n=6). Conclusions and discussion: In conclusion, PEA functionalized mimetic autografts show an important increase in bone healing, enhancing BMP-2 effects, which provide representative regeneration with a 100 folds lower dose than typically described in literature

Tissue engineered mimetic periosteum for efficient delivery of rhBMP-2

Background: Despite its unique regenerative capacity, bone healing can be compromised, leading to delayed fracture regeneration and consequently nonunion. Due to the scarcity of autografts and the problems associated with a supraphysiological use of rhBMP-2, novel tissue engineering strategies arise as a promising solution to overcome nonunions and related bone pathologies. Purpose: To clinically deal with fracture nonunion, we designed engineered mimetic autografts consisting of a personalized polycaprolactone (PCL) scaffold surrounded by a porous PCL membrane mimicking the periosteum synthesized by melt electrowriting (MEW) (Figure 1). Methods: MEW membrane was functionalized with poly ethyl acrylate (PEA) and Fibronectin for efficient rhBMP-2 binding and delivery. The regenerative capacity and therapeutic potential of these scaffolds were tested in vitro for osteoblast differentiation and vivo in a critical size femur defect in Sprague Dawley rats (n=6-7 animals/group) (ethical approval 073-20). Regenerative effects were assessed by qPCR, q-mCT and histological analysis. Non-parametric Kruskal Wallis test was used for statistical analysis. Results: We selected the two lowest dose implants (10 mg/ml, 51.94±8.84 ng and 25 mg/ml, 186.8±17.33) to assess release profile over time and for in vivo therapeutic effect. In vitro, single loading of 186 ng of rhBMP-2 allows similar differentiation potential that standard osteogenic differentiation medium where fresh rhBMP-2 was added twice weekly (Figure 2). In vivo, regarding bone regeneration, quantitative μCT analysis shows great bone healing of defects treated with rhBMP-2 at concentrations of 25 μg/ml (186 ng) and 10 μg/ml (52 ng). Control group, 6.80±2.47 mm3; 10 μg/ml BMP-2 group 19.53±4.266 mm3, *p=0.0324; 25 μg/ml BMP-2 group 24.48±11.30 mm3, **p=0.0087. In addition, histological analysis was carried out to determine the osteoconductive potential of our PCL core (Figure 3). Conclusion: In conclusion, PEA functionalized mimetic periosteum show an unpreceded increase in bone healing, greatly enhancing rhBMP-2 effects

Stimulation of 3D osteogenesis by mesenchymal stem cells using a nanovibrational bioreactor

Bone grafts are one of the most commonly transplanted tissues. However, autologous grafts are in short supply, and can be associated with pain and donor-site morbidity. The creation of tissue-engineered bone grafts could help to fulfil clinical demand and provide a crucial resource for drug screening. Here, we show that vibrations of nanoscale amplitude provided by a newly developed bioreactor can differentiate a potential autologous cell source, mesenchymal stem cells (MSCs), into mineralized tissue in 3D. We demonstrate that nanoscale mechanotransduction can stimulate osteogenesis independently of other environmental factors, such as matrix rigidity. We show this by generating mineralized matrix from MSCs seeded in collagen gels with stiffness an order of magnitude below the stiffness of gels needed to induce bone formation in vitro. Our approach is scalable and can be compatible with 3D scaffolds

Nanoscale coatings for ultralow dose BMP-2-driven regeneration of critical-sized bone defects

While new biomaterials for regenerative therapies are being reported in the literature, clinical translation is slow. Some existing regenerative approaches rely on high doses of growth factors, such as bone morphogenetic protein-2 (BMP-2) in bone regeneration, which can cause serious side effects. An ultralow-dose growth factor technology is described yielding high bioactivity based on a simple polymer, poly(ethyl acrylate) (PEA), and report mechanisms to drive stem cell differentiation and bone regeneration in a critical-sized murine defect model with translation to a clinical veterinary setting. This material-based technology triggers spontaneous fibronectin organization and stimulates growth factor signalling, enabling synergistic integrin and BMP-2 receptor activation in mesenchymal stem cells. To translate this technology, for the first time, plasma-polymerized PEA is used on 2D and 3D substrates to enhance cell signalling in vitro, showing the complete healing of a critical sized bone injury in mice in vivo. Efficacy is demonstrated in a Münsterländer dog with a nonhealing humerus fracture, establishing the clinical translation of advanced ultralow-dose growth factor treatment