Mimicking bone extracellular matrix: from BMP-2-derived sequences to osteogenic-multifunctional coatings

Cell–material interactions are regulated by mimicking bone extracellular matrix on the surface of biomaterials. In this regard, reproducing the extracellular conditions that promote integrin and growth factor (GF) signaling is a major goal to trigger bone regeneration. Thus, the use of synthetic osteogenic domains derived from bone morphogenetic protein 2 (BMP-2) is gaining increasing attention, as this strategy is devoid of the clinical risks associated with this molecule. In this work, the wrist and knuckle epitopes of BMP-2 are screened to identify peptides with potential osteogenic properties. The most active sequences (the DWIVA motif and its cyclic version) are combined with the cell adhesive RGD peptide (linear and cyclic variants), to produce tailor-made biomimetic peptides presenting the bioactive cues in a chemically and geometrically defined manner. Such multifunctional peptides are next used to functionalize titanium surfaces. Biological characterization with mesenchymal stem cells demonstrates the ability of the biointerfaces to synergistically enhance cell adhesion and osteogenic differentiation. Furthermore, in vivo studies in rat calvarial defects prove the capacity of the biomimetic coatings to improve new bone formation and reduce fibrous tissue thickness. These results highlight the potential of mimicking integrin-GF signaling with synthetic peptides, without the need for exogenous GFs.Peer ReviewedPostprint (published version

Mussel inspired chemistry and bacteria derived polymers for oral mucosal adhesion and drug delivery

Ulceration of the oral mucosa is common, can arise at any age and as a consequence of the pain lessens enjoyment and quality of life. Current treatment options often involve the use of topical corticosteroids with poor drug delivery systems and inadequate contact time. In order to achieve local controlled delivery to the lesion with optimal adhesion, we utilized a simple polydopamine chemistry technique inspired by mussels to replicate their adhesive functionality. This was coupled with production of a group of naturally produced polymers, known as polyhydroxyalkanoates (PHA) as the delivery system. Initial work focused on the synthesis of PHA using Pseudomonas mendocina CH50; once synthesized and extracted from the bacteria, the PHAs were solvent processed into films. Polydopamine coating was subsequently achieved by immersing the solvent cast film in a polymerized dopamine solution. Fourier Transform Infrared Spectroscopy (FTIR) spectroscopy confirmed functionalization of the PHA films via the presence of amine groups. Further characterization of the samples was carried out via surface energy measurements and Scanning Electron Microscopy (SEM) micrographs for surface topography. An adhesion test via reverse compression testing directly assessed adhesive properties and revealed an increase in polydopamine coated samples. To further identify the effect of surface coating, LIVE/DEAD imaging and Alamar Blue metabolic activity evaluated attachment and proliferation of fibroblasts on the biofilm surfaces, with higher cell growth in favor of the coated samples. Finally, in vivo biocompatibility was investigated in a rat model where the polydopamine coated PHA showed less inflammatory response over time compared to uncoated samples with sign of neovascularization. In conclusion, this simple mussel inspired polydopamine chemistry introduces a step change in bio-surface functionalization and holds great promise for the treatment of oral conditions

Inclusion of calcium phosphate does not further improve in vitro and in vivo osteogenesis in a novel, highly biocompatible, mechanically stable and 3D printable polymer

At a time of unpredictable challenges for health, one trend is certain: there is an exceedingly high demand for functional implants, particularly bone grafts. This has encouraged the emergence of bone tissue engineering substitutes as an alternative method to conventional bone grafts. However, the current approaches in the field face several limitations that have prevented the ultimate translation into clinical settings. As a result, many attempts have been made to fabricate synthetic bone implants that can offer suitable biological and mechanical properties.Light curable methacrylate-based polymers have ideal properties for bone repair. These materials are also suitable for 3D printing which can be applicable for restoration of both function and aesthetics. The main objective of this research was to investigate the role of calcium phosphate (CaP) incorporation in a mechanically stable, biologically functional and 3D printable polymer for the reconstruction of complex craniofacial defects. The experimental work initially involved the synthesis of (((((((((((3R,3aR,6S,6aR)- hexahydrofuro[3,2-b]furan-3,6-diyl)bis(oxy))bis(ethane-2,1- 48 diyl))bis(oxy))bis(carbonyl))bis(azanediyl))bis(3,3,5-trimethylcyclohexane-5,1- 49 diyl))bis(azanediyl))bis(carbonyl))bis(oxy))bis(ethane-2,1-diyl) bis(2-methylacrylate) referred to as CSMA and fabrication of composite discs via a Digital Light Printing (DLP) method. The flow behaviour of the polymer as a function of CaP addition, surface remineralisation potential, in vitro cell culture, using MC3T3 and Adipose-Derived Mesenchymal Stem Cells (ADSCs) and ex ovo angiogenic response was assessed. Finally, in vivo studies were carried out to investigate neo-bone formation at 4- and 8-weeks post-implantation. Quantitative micro-CT and histological evaluation did not show a higher rate of bone formation in CaP filled CSMA composites compared to CSMA itself. Therefore, such polymeric systems hold promising features by allowing more flexibility in designing a 3D printed scaffold targeted at the reconstruction of maxillofacial defects

Therapeutic Application of an Ag-Nanoparticle-PNIPAAm-Modified Eggshell Membrane Construct for Dermal Regeneration and Reconstruction

Current therapeutic treatments for the repair and/or replacement of damaged skin following disease or traumatic injury is severely limited. The chicken eggshell membrane (ESM) is a unique material: its innate physical and mechanical characteristics offer optimal barrier properties and, as a naturally derived extract, it demonstrates inherent biocompatibility/biodegradability. To further enhance its therapeutic and clinical potential, the ESM can be modified with the thermo-responsive polymer, poly(N-isopropylacrylAmide) (PNIPAAm) as well as the incorporation of (drug-loaded) silver nanoparticles (AgNP); essentially, by a simple change in temperature, the release and delivery of the NP can be targeted and controlled. In this study, ESM samples were isolated using a decellularization protocol, and the physical and mechanical characteristics were profiled using SEM, FT-IR, DSC and DMA. PNIPAAm was successfully grafted to the ESM via amidation reactions and confirmed using FT-IR, which demonstrated the distinctive peaks associated with Amide A (3275 cm−1), Amide B (2970 cm−1), Amide I (1630 cm−1), Amide II (1535 cm−1), CH2, CH3 groups, and Amide III (1250 cm−1) peaks. Confirmation of the incorporation of AgNP onto the stratified membrane was confirmed visually with SEM, qualitatively using FT-IR and also via changes in absorbance at 380 nm using UV-Vis spectrophotometry during a controlled release study for 72 h. The biocompatibility and cytotoxicity of the novel constructs were assessed using human dermal fibroblast (HDFa) and mouse dermal fibroblast (L929) cells and standard cell culture assays. Metabolic activity assessment (i.e., MTS assay), LDH-release profiles and Live/Dead staining demonstrated good attachment and spreading to the samples, and high cell viability following 3 days of culture. Interestingly, longer-term viability (>5 days), the ESM-PNIPAAm and ESM-PNIPAAm (AgNP) samples showed a greater and sustained cell viability profile. In summary, the modified and enhanced ESM constructs were successfully prepared and characterized in terms of their physical and mechanical profiles. AgNP were successfully loaded into the construct and demonstrated a desirable release profile dependent on temperature modulation. Fibroblasts cultured on the extracted ESM samples and ESM-PNIPAAm demonstrated high biocompatibility in terms of high cell attachment, spreading, viability and proliferation rates. As such, this work summarizes the development of an enhanced ESM-based construct which may be exploited as a clinical/therapeutic wound dressing as well as a possible application as a novel biomaterial scaffold for drug development

Hyperelastic, shape‐memorable, and ultra‐cell‐adhesive degradable polycaprolactone‐polyurethane copolymer for tissue regeneration

Novel polycaprolactone-based polyurethane (PCL-PU) copolymers with hyperelasticity, shape-memory, and ultra-cell-adhesion properties are reported as clinically applicable tissue-regenerative biomaterials. New isosorbide derivatives (propoxylated or ethoxylated ones) were developed to improve mechanical properties by enhanced reactivity in copolymer synthesis compared to the original isosorbide. Optimized PCL-PU with propoxylated isosorbide exhibited notable mechanical performance (50 MPa tensile strength and 1150% elongation with hyperelasticity under cyclic load). The shape-memory effect was also revealed in different forms (film, thread, and 3D scaffold) with 40%–80% recovery in tension or compression mode after plastic deformation. The ultra-cell-adhesive property was proven in various cell types which were reasoned to involve the heat shock protein-mediated integrin (α5 and αV) activation, as analyzed by RNA sequencing and inhibition tests. After the tissue regenerative potential (muscle and bone) was confirmed by the myogenic and osteogenic responses in vitro, biodegradability, compatible in vivo tissue response, and healing capacity were investigated with in vivo shape-memorable behavior. The currently exploited PCL-PU, with its multifunctional (hyperelastic, shape-memorable, ultra-celladhesive, and degradable) nature and biocompatibility, is considered a potential tissue- regenerative biomaterial, especially for minimally invasive surgery that requires small incisions to approach large defects with excellent regeneration capacity

Intra-articular biomaterials-assisted delivery to treat temporomandibular joint disorders

The temporomandibular joint disorder, also known as myofascial pain syndrome, is considered one of the prevalent chronic pain diseases caused by muscle inflammation and cartilage degradation in head and neck, and thus influences even biopsychosocial conditions in a lifetime. There are several current treatment methodologies relieving inflammation and preventing degradation of the joint complex. One of the promising non-surgical treatment methods is an intra-articular injection of drugs such as corticosteroids, analgesics, and anti-depressants. However, the side effects of drugs due to frequent injections and over-doses, including dizziness, dry mouth, and possible drug dependency are considered limitations. Thus, the delivery of therapeutic molecules through the use of nano/microparticles is currently considered as a promising strategy primarily due to the controlled release. This review highlights the nano/microparticle systems for effective intra-articular therapeutics delivery to prevent cartilage degradation and protect subchondral bone in a temporomandibular joint

Multifunctional dendrimer@nanoceria engineered GelMA hydrogel accelerates bone regeneration through orchestrated cellular responses

Bone defects in patients entail the microenvironment that needs to boost the functions of stem cells (e.g., proliferation, migration, and differentiation) while alleviating severe inflammation induced by high oxidative stress. Biomaterials can help to shift the microenvironment by regulating these multiple events. Here we report multifunctional composite hydrogels composed of photo-responsive Gelatin Methacryloyl (GelMA) and dendrimer (G3)-functionalized nanoceria (G3@nCe). Incorporation of G3@nCe into GelMA could enhance the mechanical properties of hydrogels and their enzymatic ability to clear reactive oxygen species (ROS). The G3@nCe/GelMA hydrogels supported the focal adhesion of mesenchymal stem cells (MSCs) and further increased their proliferation and migration ability (vs. pristine GelMA and nCe/GelMA). Moreover, the osteogenic differentiation of MSCs was significantly stimulated upon the G3@nCe/GelMA hydrogels. Importantly, the capacity of G3@nCe/GelMA hydrogels to scavenge extracellular ROS enabled MSCs to survive against H2O2-induced high oxidative stress. Transcriptome analysis by RNA sequencing identified the genes upregulated and the signalling pathways activated by G3@nCe/GelMA that are associated with cell growth, migration, osteogenesis, and ROS-metabolic process. When implanted subcutaneously, the hydrogels exhibited excellent tissue integration with a sign of material degradation while the inflammatory response was minimal. Furthermore, G3@nCe/GelMA hydrogels demonstrated effective bone regeneration capacity in a rat critical-sized bone defect model, possibly due to an orchestrated capacity of enhancing cell proliferation, motility and osteogenesis while alleviating oxidative stress

Effect of lyophilized gelatin-norbornene cryogel size on calvarial bone regeneration

Molding processes with molds containing topographical structures have been used for fabrication of hydrogel and cryogel particles. However, they can involve difficulties in separation of fabricated particles with complex shape from the molds or repeated fabrication of the particles although the overall processes do not require much skill and equipment. In this study, molds with etched superhydrophobic patterns have been developed by etching polytetrafluoroethylene (PTFE) blocks in user-defined designs with a femtosecond (FS) laser-based etching system. Lyophilized cryogel particles with various designs and sizes were fabricated by molding precursors with these PTFE molds. Additionally, the clean and easy separation of particles from the molds allowed repeated fabrication of the particles. For an application, relatively ‘big’ gelatin-norbornene (GelNB) cryogel particles prepared via molding with polydimethylsiloxane (PDMS) molds, swelling in phosphate buffered saline (PBS) and slicing height in half and ‘small’ GelNB cryogel particles fabricated with the PTFE molds were fabricated. Then, they were used to study scaffold size effect on calvarial bone regeneration. The molds generated with the FS laser-based etching system can be useful for various applications that require the mass production of cryogel particles in various geometries

Characterization of Physical and Biological Properties of a Caries-Arresting Liquid Containing Copper Doped Bioglass Nanoparticles

Silver diamine fluoride (SDF) is an outstanding dental material for arresting and preventing caries, but some drawbacks, such as high flowability due to low viscosity and cytotoxicity to the pulp, have been reported. To overcome these problems, copper-doped bioactive glass nanoparticles (CuBGns) were combined with SDF. After synthesis, CuBGns were examined by physical analysis and added in SDF at different weight/volume% (SDF@CuBGn). After assessing physical properties (viscosity and flowability) of SDF@CuBGn, physicochemical properties (morphology before and after simulated body fluid (SBF) immersion and ion release) of SDF@CuBGn-applied hydroxyapatite (HA) discs were evaluated. Biological properties were further evaluated by cytotoxicity test to pulp stem cells and antibacterial effect on cariogenic organisms (Streptococcus mutans and Staphylococcus aureus). Combining CuBGns in SDF increased the viscosity up to 3 times while lowering the flowability. More CuBGns and functional elements in SDF (Ag and F) were deposited on the HA substrate, even after SBF immersion test for 14 days, and they showed higher Cu, Ca, and Si release without changing F and Ag release. Cell viability test suggested lower cytotoxicity in SDF@CuBGn-applied HA, while CuBGns in SDF boosted antibacterial effect against S. aureus, ~27% in diameter of agar diffusion test. In conclusion, the addition of CuBGn to SDF enhances viscosity, Ag and F deposition, and antibacterial effects while reducing cell toxicity, highlighting the role of bioactive CuBGns for regulating physical and biological effects of dental materials