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CLSM images presenting immunofluorescent (IF) staining of type I collagen and osteocalcin in the extracellular matrix (ECM) of MSCs cultured in PS wells in the presence of scaffolds extracts (control medium, cells maintained in osteogenic medium); collagen, red fluorescence; osteocalcin, green fluorescence; and nuclei, blue fluorescence, magnified 400×; scale bar = 50 µm.
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![Mg 2+ and Zn 2+ ion concentrations [µg/mL] in scaffold extracts...](publication/335018042/figure/tbl2/AS:789249146241030@1565183068568/Mg-2-and-Zn-2-ion-concentrations-g-mL-in-scaffold-extracts-prepared-by-24-h_Q320.jpg)
Modification of implantable scaffolds with magnesium and zinc for improvement of bone regeneration is a growing trend in the engineering of biomaterials. The aim of this study was to synthesize nano-hydroxyapatite substituted with magnesium (Mg2+) (HA-Mg) and zinc (Zn2+) (HA-Zn) ions in order to fabricate chitosan-agarose-hydroxyapatite (HA) scaffo...
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... reduced level of bALP and OC, along with poor ECM calcification, suggests that ADSCs cultured in the extracts containing Mg 2+ and/or Zn 2+ were still in the first stage of osteogenic differentiation. Unlike cells exposed to the scaffold extracts, the ADSCs cultured in the control medium showed a moderate bALP level (Figure 7b), high OC production, and high mineralization activity (Figures 8 and 9), and therefore they were in the third phase of osteogenic differentiation. ...
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Citations
... The effect of incorporating zinc ions into magnesium-doped nanohydroxyapatite-based biomaterials remains controversial. Kazimierczak et al. 130 investigated the addition of magnesium and zinc ions to chitosan−agarose−hydroxyapatite scaffolds but did not observe any enhancement in biological properties compared to the sole addition of zinc ions. They proposed that including zinc ions in polymer scaffolds based on Mg-doped nanohydroxyapatite did not yield favorable effects on cellular behavior. ...
Ion substitution techniques for nanoparticles have become an important neighborhood of biomedical engineering and have led to the development of innovative bioactive materials for health systems. Magnesium-doped nanohydroxyapatite (Mg-nHA) has good bone conductivity, biological activity, flexural strength, and fracture toughness due to particle doping technology, making it an ideal candidate material for biomedical applications. In this Review, we have systematically presented the synthesis methods of Mg-nHA and their application in the field of biomedical science and highlighted the pros and cons of each method. Finally, some future prospects for this important neighborhood are proposed. The purpose of this Review is to provide readers with an understanding of this new field of research on bioactive materials with innovative functions and systematically introduce the latest technologies for obtaining uniform, continuous, and morphologically diverse Mg-nHA.
... Additionally, it provided a favorable extracellular matrix microenvironment for in situ osteogenesis and accelerated bone tissue regeneration in rat calvarial bone defects. 115 To improve the stability of magnesium ions, researchers often choose other components, with HA being the Mg 2+ -substituted nHA and prepared a chitosan-agarose HA gel with improved biocompatibility. Incorporating Mg 2+ into these structures can significantly improve the biocompatibility of the resulting materials, thereby increasing their potential for biomedical applications. ...
... A network structure or crosslinked network can also be formed in a scaffold to develop self-healing polymers that can repair cracks and fractures in the material. 114,115 Alternatively, shape memory polymers can be used because of their ability to return to their original shape after deformation. 124 The primary advantage of composite materials is their biodegradability; however, the application of these materials needs to be adjusted according to the complex environment of different tissues. ...
The increasing prevalence of orthopedic‐related diseases necessitates the development of effective orthopedic implants. Conventional metal scaffolds used in orthopedic devices have limitations such as poor biocompatibility and the need for a second surgery to remove the scaffold. Degradable metal‐based scaffolds, including metal‐based scaffolds and multifunctional scaffolds doped with metal elements, are a rapidly developing tissue engineering strategy aimed at utilizing the mechanical and biological properties of metal elements to create a support structure that matches the complex bone regeneration environment. Repairing bone defects involves the regeneration of various tissues, and incomplete repair can negatively affect bone function and overall recovery. Therefore, combining metal‐based degradable materials with other active components has great potential for enhancing the versatility and clinical applications of scaffolds. Multifunctional scaffolds doped with metal elements have better biocompatibility, osteoinductivity, biodegradability, matching mechanical, and microenvironmental adjustment capabilities. Moreover, these metal‐doped scaffolds possess the advantages of controlled release of metal ions, multifunctionality, and faster degradation. This review focuses on the materials and techniques used for constructing degradable metal‐based scaffolds. Furthermore, this study discussed the potential for designing and constructing multifunctional biodegradable metal‐based scaffolds doped with metal elements by integrating multiple strategies.
... Kazimierczak et al. have synthesized magnesium (HA-Mg) and (HA-Zn) ion-substituted nano-hydroxyapatite (HA) synthesized to prepare a more biocompatible chitosanagarose-hydroxyapatite (HA) scaffold (chit/aga/HA). 112 These two above-mentioned metal ions have been found to have synergistic effects on the biological response in cell tests. The addition of Mg 2+ to this biomaterial structure can promote osteoblast spreading, promote cell proliferation on the scaffold surface, and promote osteocalcin production by mesenchymal stem cells. ...
Biomaterial implantation into the human body plays a key role in the medical field and biological applications. Increasing the life expectancy of biomaterial implants, reducing the rejection reaction inside the human body and reducing the risk of infection are the problems in this field that need to be solved urgently. The surface modification of biomaterials can change the original physical, chemical and biological properties and improve the function of materials. This review focuses on the application of surface modification techniques in various fields of biomaterials reported in the past few years. The surface modification techniques include film and coating synthesis, covalent grafting, self-assembled monolayers (SAMs), plasma surface modification and other strategies. First, a brief introduction to these surface modification techniques for biomaterials is given. Subsequently, the review focuses on how these techniques change the properties of biomaterials, and evaluates the effects of modification on the cytocompatibility, antibacterial, antifouling and surface hydrophobic properties of biomaterials. In addition, the implications for the design of biomaterials with different functions are discussed. Finally, based on this review, it is expected that the biomaterials have development prospects in the medical field.
... After 24 h incubation at 37 • C, the culture medium was replaced with 500 µL of osteogenic medium containing essential oil-derived compounds, and the cells were cultured as described above. After a 21-day exposure of MC3T3-E1 cells and ADSCs to the osteogenic medium supplemented with tested compounds, qualitative and quantitative evaluation of the mineral deposition in ECM was conducted by Alizarin Red S staining, according to the procedure described in our previous study [47]. In brief, cells were fixed with 3.7% paraformaldehyde, rinsed twice with PBS, and stained for 15 min at 37 • C with a 2% (w/v) ARS (all reagents were purchased from Sigma-Aldrich Chemicals, Warsaw, Poland) solution prepared in deionized water. ...
... The mineral deposits in the ECM were visualized by a stereoscopic microscope (Olympus SZ61TR, Olympus Polska Sp. z o. o., Warsaw, Poland). After visualization, the same samples were subjected to quantitative evaluation of the mineral deposits in the ECM according to the procedure described earlier [47]. The calcium-bound ARS was dissolved by using 10% acetic acid (Sigma-Aldrich Chemicals, Warsaw, Poland). ...
Over the years, essential oils (EOs) and their compounds have gained growing interest due to their anti-inflammatory, antimicrobial, antioxidant, and immunomodulatory properties. The aim of this study was to evaluate the effect of eight commercially available EO-derived compounds ((R)-(+)-limonene, (S)-(−)-limonene, sabinene, carvacrol, thymol, alpha-pinene (α-pinene), beta-pinene (β-pinene), and cinnamaldehyde) on the bone formation process in vitro to select the most promising natural agents that could potentially be used in the prevention or treatment of osteoporo-sis. Within this study, evaluation of cytotoxicity, cell proliferation, and osteogenic differentiation was performed with the use of mouse primary calvarial preosteoblasts (MC3T3-E1). Moreover, extracellular matrix (ECM) mineralization was determined using MC3T3-E1 cells and dog adipose tissue-derived mesenchymal stem cells (ADSCs). The two highest non-toxic concentrations of each of the compounds were selected and used for testing other activities. The conducted study showed that cinnamaldehyde, thymol, and (R)-(+)-limonene significantly stimulated cell proliferation. In the case of cinnamaldehyde, the doubling time (DT) for MC3T3-E1 cells was significantly shortened to approx. 27 h compared to the control cells (DT = 38 h). In turn, cinnamaldehyde, carvacrol, (R)-(+)-limonene, (S)-(−)-limonene, sabinene, and α-pinene exhibited positive effects on either the synthesis of bone ECM or/and mineral deposition in ECM of the cells. Based on the conducted research, it can be assumed that cinnamaldehyde and (R)-(+)-limonene are the most promising among all tested EO-derived compounds and can be selected for further detailed research in order to confirm their biomedical potential in the chemoprevention or treatment of osteoporosis since they not only accelerated the proliferation of preosteoblasts, but also significantly enhanced osteocalcin (OC) synthesis by preosteoblasts (the OC level was approx. 1100-1200 ng/mg compared to approx. 650 ng/mg in control cells) and ECM calcification of both preosteoblasts and mesenchymal stem cells. Importantly, cinnamaldehyde treatment led to a threefold increase in the mineral deposition in ADSCs, whereas (R)-(+)-limonene caused a twofold increase in the ECM mineralization of both MC3T3-E1 cells and ADSCs.
... The presence of Ca 2+ provided enhancement effects on cell adhesion, cell proliferation, and cell differentiation. In addition, the released PO 4 3− ions provided an essential effect by regulating cell proliferation and stimulating the proteins related to ECM mineralization [67]. According to these data, the positive effect of the composite PDA-assisted biomineralized layer on the osteogenic improvement of the developed porous PLA/PBAT scaffold was confirmed. ...
The development of scaffolds that simultaneously provide porous architectures and osteogenic properties is the major challenge in tissue engineering. Herein, a scaffold with high porosity and well interconnected networks, namely poly(lactic acid)/poly(butylene adipate-co-terephthalate) (PLA/PBAT), was fabricated using the gas foaming/ammonium bicarbonate particulate leaching technique. Mussel-inspired polydopamine (PDA)-assisted biomineralization generated by two-step simple soaking in dopamine solution and 10× SBF-like solution was performed to improve the material’s osteogenicity. Highly porous scaffolds available in less organized opened cell structures with diameters ranging from 10 µm to 100 µm and 200 µm to 500 µm were successfully prepared. The well interconnected porous architectures were observed through the whole thickness of the scaffold. The even deposition of the organic–inorganic bioactive mineralized layer composed of PDA and nano-scale hydroxyapatite (HA) crystals on the scaffold surface was evidenced by scanning electron microscopy (SEM), energy-dispersive X-ray analysis (EDX), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). The developed scaffold exhibited high total porosity (84.17 ± 1.29%), a lower surface contact angle (θ = 45.7 ± 5.9°), lower material degradation rate (7.63 ± 2.56%), and a high level of material biocompatibility. The MTT assay and Alizarin Red S staining (ARS) confirmed its osteogenic enhancement property toward human osteoblast-like cells (MG-63). These results clarified that the developed porous PLA/PBAT scaffold with PDA-assisted biomineralization exhibited good potential for application as a biomaterial for bone tissue regeneration and hard tissue engineering.
... It is worth underlining that osteoblast differentiation is a complex, three-stage process, which may be regulated by composition of biomaterials as well as their structural, physicochemical, and mechanical properties [21,38,54]. In brief, at the beginning of differentiation, the osteoblasts mainly synthesize collagen I. ...
... Then, during the second stage of differentiation, the cells secrete high amounts of bALP. In turn, at the end of this process, the level of bALP decreases and osteoblasts produce a great amount of proteins involved in calcification of ECM (e.g., osteocalcin) [21,38,54]. Considering all the results obtained in this study (Figures 10a-c and 11), it may be stated that both PS (control) and the biomaterials allowed proper differentiation of human osteoblasts. ...
The number of bone fractures and cracks requiring surgical interventions increases every
year; hence, there is a huge need to develop new potential bone scaffolds for bone regeneration. The goal of this study was to gain knowledge about the basic properties of novel curdlan/whey protein isolate/hydroxyapatite biomaterials in the context of their use in bone tissue engineering. The purpose of this research was also to determine whether the concentration of whey protein isolate in scaffolds has an influence on their properties. Thus, two biomaterials differing in the concentration of whey protein isolate (i.e., 25 wt.% and 35 wt.%; hereafter called Cur_WPI25_HAp and Cur_WPI35_HAp, respectively) were fabricated and subjected to evaluation of porosity, mechanical properties, swelling ability, protein release capacity, enzymatic biodegradability, bioactivity, and cytocompatibility towards osteoblasts in vitro. It was found that both biomaterials fulfilled a number of requirements for bone scaffolds, as they demonstrated limited swelling and the ability to undergo controllable enzymatic biodegradation, to form apatite layers on their surfaces and to support the viability, growth, proliferation, and differentiation of osteoblasts. On the other hand, the biomaterials were characterized by low open porosity, which may hinder the penetration of cells though their structure. Moreover, they had low mechanical properties compared to natural bone, which limits their use to filling of bone defects in non-load bearing implantation areas, e.g., in the craniofacial area, but then they will be additionally supported by application of mechanically strong materials such as titanium plates. Thus, this preliminary in vitro research indicates that biomaterials composed
of curdlan, whey protein isolate, and hydroxyapatite seem promising for bone tissue engineering applications, but their porosity and mechanical properties should be improved. This will be the subject of our further work.
... The scaffolds were fabricated by previously developed and patented (Polish Patent no. 235822) new method combining foaming agent with freeze-drying technique to obtain highly macroporous structure of the resultant material [21,22]. Within this study, reliable microbiological tests were performed according to OECD Document No. 202, JT03360420 to evaluate bactericidal and bacteriostatic. ...
... Biomaterials were prepared in accordance with the procedure described in the Polish Patent no. 235822 and as described previously [21,22]. Briefly, the suspension of 2 wt% chitosan (50-190 kDa molecular weight, 75-85 % deacetylation degree, viscosity ≤300 cP, Sigma-Aldrich Chemicals, Poland) and 5 wt% agarose (gel point 36 ± 1.5 • C, low EEO, Sigma-Aldrich Chemicals, Poland) in 2 % (v/v) acetic acid solution (Avantor Performance Materials, Poland) was mixed with 40 wt% HA or Zn-doped HA nanopowder. ...
... Moreover, in our previous study, it was proven that mat_HA/Zn-0.03 was favorable to the proliferation of mouse calvarial preosteoblasts (MC3T3-E1 Subclone 4) and enhanced type I collagen production and extracellular matrix mineralization in human bone marrow-derived mesenchymal stem cells (BMDSCs) during long-term experiments [22]. Thus, mat_HA/Zn-0.03 is the most appropriate for biomedical applications to support bone regeneration and prevent post-surgery infections as it not only provides antibacterial properties, but also is nontoxic against eukaryotic cells. ...
Development of bone scaffolds that are nontoxic to eukaryotic cells, while revealing bactericidal activity still remains a huge challenge for the scientific community. It should be noted that only bacteriostatic (the ability of the biomaterial to inhibit the growth of bacteria) and bactericidal (the ability to kill >99.9 % bacteria) activities have clinical importance. Unfortunately, many material scientists are confused with the microbiological definition of antibacterial action and consider biomaterials causing reduction in colony-forming units (CFUs) by 50–80 % as promising antibacterial implants. The aim of this study was to synthesize three variants of Zn-doped hydroxyapatite (HA) nanopowder, which were characterized by different content of Zn²⁺ and served as a powder phase for the production of novel macroporous chitosan/agarose/nanoHA biomaterials with high antibacterial activity. Within this study, it was proven that the scaffold with a low zinc content (doping level 0.03 mol for 1 mol of HA; 0.2 wt%) revealed the gradual and slow release of the Zn²⁺ ions, preventing against accumulation of high and toxic concentration of therapeutic agents and providing prolonged antibacterial activity. Moreover, developed biomaterial was nontoxic to human osteoblasts and showed anti-biofilm properties, bactericidal activity (> 99.9 % of bacteria killed) against Staphylococcus epidermidis and Escherichia coli, significant antibacterial activity against Staphylococcus aureus (98.5 % of bacteria killed), and also bacteriostatic activity against Pseudomonas aeruginosa. Thus, the developed Zn-doped HA-based bone scaffold has excellent antibacterial properties without toxicity against eukaryotic cells, being a promising biomaterial for biomedical applications to repair bone defects and prevent post-surgery infections.
... To obtain bioactive compound-loaded biomaterials, curdlan/agarose water suspension was additionally mixed with 0.2 % (w/v) gentamicin sulfate salt (Merck, Warsaw, Poland) or 4 % (w/v) zinc-doped nano-hydroxyapatite powder with doping level 1 mol for 1 mol of hydroxyapatite (theoretical zinc content = 6.4 wt%; performed ICP-OES analysis confirmed 4.9 wt% zinc content). The zinc-doped nano-hydroxyapatite powder was synthesized according to the procedure described by Kazimierczak et al. [28]. Before all experiments, the biomaterials were sterilized by ethylene oxide. ...
A topical application of antibiotic-loaded wound dressings is recommended only for chronically infected wounds with poor vascularization. Thus, more often dressing materials loaded with antibacterial metal ions are produced. In turn, gentamicin sponges are commonly used to prevent surgical site infections. The aim of this study was to produce curdlan-based biomaterials enriched with gentamicin and zinc (Zn)-doped nano-hydroxyapatite to prevent wound and surgical site infections. Developed biomaterials were subjected to basic microstructural characterization, cytotoxicity test against human skin fibroblasts (BJ cell line), and comprehensive microbiological experiments using Staphylococcus aureus and Pseudomonas aeruginosa strains. To evaluate the in vivo healing capacity of the developed biomaterials, severely infected chronic wound in a veterinary patient was treated with the use of gentamicin-loaded dressing.
Fabricated biomaterials were characterized by a highly porous microstructure with high plasma absorption capacity (approx. 7 mL/g for Zn-loaded biomaterial and 13 mL/g for gentamicin-enriched dressing) and optimal water vapor transmission rate (approx. 1700 g/m²/day). Due to the presence of bioceramics, material containing Zn showed slightly higher compressive strength (0.37 MPa) and Young's modulus (3.33 MPa) values compared to gentamicin-loaded biomaterial (0.12 MPa and 1.29 MPa, respectively). Gentamicin-enriched biomaterial showed burst release of the drug within the first 5 h, while, the zinc-loaded biomaterial exhibited a constant gradual release of the zinc ions. Conducted assays showed that developed biomaterials were non-toxic against human skin fibroblasts (cell viability in the range of 71–95 %) and revealed strong bactericidal activity (99.9 % reduction in the number of viable bacterial CFUs in direct contact test) against S. aureus. In the case of P. aeruginosa, only gentamicin-loaded biomaterial exhibited bactericidal effect. Additionally, biomaterials had the ability to uptake, lock in, and kill bacteria within their gel structure, enabling the cleansing of the wound bed at every dressing change. Finally, the treatment of severely infected wound in veterinary patient confirmed the effectiveness of gentamicin-loaded biomaterial.
Biomaterial enriched with gentamicin possesses great potential to be used as a dressing material or sponge for the treatment of chronically infected wounds and surgical site infections. In turn, the zinc-loaded biomaterial may be used as a wound dressing to reduce and prevent microbial contamination.
... CS has attracted considerable attention in bone tissue engineering, owing to its innate antimicrobial, osteo-inductive, and mechanical properties. CS-based topical drug delivery systems can promote bone regeneration and treat bone diseases, with CS showing promising efficacy in OM treatment (Kazimierczak et al., 2019;Kimna et al., 2019;Tao J. et al., 2020). In addition to antibiotics (Li et al., 2020a;Tao J. et al., 2020), silver can also be released in a sustained manner from CS hydrogel in vivo (Croes et al., 2018). ...
Osteomyelitis (OM), a devastating disease caused by microbial infection of bones, remains a major challenge for orthopedic surgeons. Conventional approaches for prevention and treatment of OM are unsatisfactory. Various alternative strategies have been proposed, among which, hydrogel-based strategies have demonstrated potential due to their unique properties, including loadable, implantable, injectable, printable, degradable, and responsive to stimuli. Several protocols, including different hydrogel designs, selection of antimicrobial agent, co-administration of bone morphogenetic protein 2 (BMP 2), and nanoparticles, have been shown to improve the biological properties, including antimicrobial effects, osteo-induction, and controlled drug delivery. In this review, we describe the current and future directions for designing hydrogels and their applications to improve the biological response to OM in vivo.
... Cell adhesion is a critical factor for cell proliferation on the scaffold surface 66 . SEM analysis clearly show that MSC efficiently adhere to the scaffold surface, and gradually change their cell morphology, from a spindle-like shape with round nuclei at day 7 (Fig. 4, indicated by an arrow) to an elongated shape with an increased number of cellular extensions after 14 and 21 days of culture. ...
... The second differentiation stage of MSC involves ECM synthesis, when cells exhibit the highest alkaline phosphatase (ALP) activity. ALP contributes to the initiation of osteoblast mineralization, at the early stages, catalyzing the hydrolysis of organic phosphate to inorganic phosphate, which is a key factor for mineral deposition 66,67 . The effects of the 3D-printed scaffolds on ALP activity were evaluated after incubation of MSC with the scaffolds for 7, 14 or 21 days under growth or osteogenic differentiation conditions (Fig. S9). ...
Bone defects stand out as one of the greatest challenges of reconstructive surgery. Fused deposition modelling (FDM) allows for the printing of 3D scaffolds tailored to the morphology and size of bone damage in a patient-specific and high-precision manner. However, FDM still suffers from the lack of materials capable of efficiently supporting osteogenesis. In this study, we developed 3D-printed porous scaffolds composed of polylactic acid/hydroxyapatite (PLA/HA) composites with high ceramic contents (above 20%, w/w) by FDM. The mechanical properties of the PLA/HA scaffolds were compatible with those of trabecular bone. In vitro degradation tests revealed that HA can neutralize the acidification effect caused by PLA degradation, while simultaneously releasing calcium and phosphate ions. Importantly, 3D-printed PLA/HA did not induce the upregulation of activation markers nor the expression of inflammatory cytokines in dendritic cells thus exhibiting no immune-stimulatory properties in vitro. Evaluations using human mesenchymal stem cells (MSC) showed that pure PLA scaffolds exerted an osteoconductive effect, whereas PLA/HA scaffolds efficiently induced osteogenic differentiation of MSC even in the absence of any classical osteogenic stimuli. Our findings indicate that 3D-printed PLA scaffolds loaded with high concentrations of HA are most suitable for future applications in bone tissue engineering.
