Biomimetic Polyurethanes in Tissue Engineering

5.Skeletal Muscle Tissue Engineering

Skeletal muscles are the major muscles in our body and have various critical functions [84].

Skeletal muscles make up to 40% of the total body mass. Not only do they play a key role in our movements, but they also keep our bodies in a state of balance by regulating energy production. One of the most important physiological features that help maintain this balance is the extraordinary ability of skeletal muscles to adapt to changed functional needs. On a macro scale, such adaptation is clearly seen in athletes, marathon runners and weightlifters. While marathon runners' training focuses on strengthening fatigue-resistant muscles, weightlifters' training focuses on working on muscles that react quickly and produce a lot of power. A number of disorders can severely impair normal muscle function. In particular, traumatic skeletal muscle injury can be caused by bruising, extreme cold or toxins, road accidents, surgery, or sports-related injuries [85]. Up to 55% of all sports injuries result in damage at the muscle fiber level [86]. Loss of more than 20% of total muscle mass results in reduced function and impaired tissue regeneration [87]. Despite the impressive ability of the body to regenerate on a daily basis, in some cases skeletal muscle cannot be fully rebuilt. In the case of minor damage, the muscle can recover completely, but when the damage is significant, repair of large losses of muscle mass may be impossible and the damage is irreversible. This makes rebuilding and improving muscle function one of the greatest biomedical challenges of our time. Tissue-engineered muscle represents a suitable strategy for replacing damaged or lost muscle tissue. A particular challenge, however, is producing structures that accurately mimic the mechanical properties of muscle, such as soft and flexible woven fabrics that can withstand large uniaxial loads and stretch up to 60% before mechanical failure [88].

5.1.Polyurethane Scaffolds for Skeletal Muscle Engineering

In order to design scaffolds that mimic the mechanical properties of skeletal muscle, different PU formulations were investigated as skeletal muscle scaffolds obtained by different techniques, i.e. freeze-drying, bioprinting, decellularization, and electrospinning [84].

Chen et al. [89,90] synthesized biodegradable, electroactive and elastic polyurethane-urea copolymers from amine capped aniline trimer, dimethylol propionic acid, polylactide, and hexamethylene diisocyanate. The high breaking elongation rate of these poly(urethane urea) (PUU) copolymers (between 145.2% and 641.7%) is attributed to the hydrogen bonds between the ester, urethane, and urea groups which serve as physical crosslinking points to enhance the intra- and intermolecular interaction between the PUU macromolecules. These materials have also been shown to have an enhanced effect on myogenic differentiation of C2C12 myoblasts.

Along this line of interest, Xu et al. [91] reported the incorporation of aniline trimer into polycaprolactonediol-based polyurethane (CPU) and then doped with camphorsulfonic acid (CSA). The electrical conductivity was studied under both dry and wet conditions. The results showed that wet CPU films exhibited better conductivity compared to dry film samples. Increasing the CSA content in the films also increased the electrical conductivity of the obtained materials.

Random block polyurethanes (PULA-ran-3/4HB) and alternating block polyurethanes (PULA-alt-3/4 HB) based on the polyester poly(lactic acid) (PLA) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3/4HB) copolymer were studied by Niu [92]. The porous PULA-alt-3/4 HB scaffolds showed hemocompatibility, in vitro and in vivo cytocompatibility, and hydrophilicity due to a more regular structure compared to PULA-ran-3/4 HB materials, making them a better candidate for muscle tissue scaffolds. Implantation in rats for six weeks was successful, and no inflammation associated with muscle tissue formation was observed in the case of alternating and random copolymeric scaffold structures.

In recent years, significant progress has been observed in electrospinning techniques, which make it possible to obtain scaffolds in the form of biomimetic nanofibrous morphologies consisting of fibers similar to fibrous ECM proteins. Such topography affects cell proliferation, adhesion and phenotype [93,94,95].

The electrospinning technique was used by Yıldırım et al. [84], who obtained nanofibers based on thermoplastic polyurethane (TPU) doped with clinoptilolite (CLN). Mechanical tests showed that pure TPU and TPU doped with 5 and 10 wt.% CLN showed Young's modulus values ​​of 3.66, 2.37 and 1.85 MPa, respectively, while the elongation at break was 118.37%, 106.18% and 179.0%, respectively. Cytotoxicity tests showed that the obtained TPU/CLN membranes are biocompatible, and cell adhesion increases proportionally with the increase in the amount of CLN in the composite.

The artificial muscles based on electrospun PU microfiber yarns, in which carbon nanotubes (CNTs) were embedded, have been studied by Meng et al. [96]. The presence of nanotubes in the yarns allows them to efficiently absorb near-infrared light and heat, which induces the fast temperature change that leads to the contraction/expansion motions along the axial direction. The yarns from microfiber mats are highly twisted into the coiled structure, which generates maximum contractive actuation of 6.7% at 70°C. 1000 cycles were carried out with no significant drop in performance.

Solution blow spinning (SBS) technique was used to produce fibrous polyurethanes with fiber diameters of 200, 500 and 1000 nm [97]. The effect of collector rotation speed on fiber alignment and diameter was investigated, and it was shown that the larger the fiber diameter, the better controlled architectures can be obtained and that the scaffold pore size increased with increasing fiber diameter, but decreased with increasing fiber alignment. Mechanical property studies showed that they strongly depend on the stretching direction, but fiber orientation affects mechanical strength only in the case of materials with a fiber diameter of 1000 nm. On scaffolds with aligned fibers and the largest fiber diameter (1000 nm), pericyte growth occurred. Scaffolds obtained by the SBS technique can be used for controlled design and production of scaffolds with aligned fibers [98].

Traditional methods such as electrospinning or freeze-drying have limitations, especially when it comes to producing realistic, large-scale muscle grafts. These techniques often require secondary colonization to obtain larger constructs, which complicates the even distribution of cells and makes it difficult to ensure a homogeneous population of different cell types. Among the various available fabrication techniques, 3D bioprinting stands out for its precision in creating the complex architecture required for efficient skeletal muscle. This method allows for the deposition of cell-laden hydrogels in a controlled, layer-by-layer manner, effectively mimicking the extracellular matrix (ECM) environment [99,100,101].

The study by Gokyer et al. [102] focused on the development and evaluation of a new 3D-printed scaffold made of thermoplastic polyurethane-urea (TPU) elastomer that was designed to mimic the mechanical properties of skeletal muscle extracellular matrix (ECM). This was particularly important due to the limitations of other synthetic biomaterials such as polycaprolactone (PCL), which showed significantly reduced elasticity (28% elongation) compared to the TPU scaffold (940% elongation). The TPU scaffold was designed with a parallel internal architecture to improve cellular behavior – Figure 5. In vitro experiments demonstrated successful seeding and differentiation of C2C12 myoblasts and human adipose-derived stem cells (hADSCs) on the scaffold, a crucial step before moving on to in vivo applications.

In subsequent experiments using a rat model of volume muscle loss (VML), Gokyer et al. [102] compared the performance of cell-free scaffolds with that of cell-loaded scaffolds containing hADSCs. The results showed that the cell-loaded scaffolds promoted significant muscle tissue regeneration, while the acellular scaffolds did not show similar results. Furthermore, the study showed that the regenerated muscle tissue was able to generate force exceeding preoperative levels by 112%, which highlights the high potential of the TPU scaffold in muscle tissue engineering applications. Overall, the results of this study highlight the advantages of using TPU over traditional biomaterials for muscle regeneration, especially in terms of appropriate mechanical properties and enhanced bioactivity in combination with appropriate cell types. This work represents a significant step forward in the field of regenerative medicine and muscle tissue engineering, offering promising opportunities for future research and clinical applications.

5.2.Polyurethane Scaffolds for Cardiac Muscle Engineering

Cardiac patches, as an innovative solution in heart regeneration, play a key role in the therapy of myocardial injuries. Made of various materials, both natural and synthetic, they aim to mimic the natural structure of heart tissue. Thanks to the microstructure that mimics the biological environment, these patches can support cell adhesion and proliferation, which promotes tissue regeneration. Adapting the patch to specific needs, such as size, shape and mechanical strength, is extremely important to ensure its compatibility with the surrounding tissues. Also, the possibility of seeding cells in the patches before their implantation opens up new possibilities in the context of heart regeneration, allowing their development and differentiation in a controlled culture environment. It is crucial that these materials can degrade in sync with the natural healing process, helping to avoid problems such as the formation of a fibrous capsule that could lead to a chronic inflammatory response [109]

Adapting the mechanical properties of materials to the characteristics of the heart is important because the stiffness of cardiac cells changes during the cardiac cycle. Stiffness values ​​that start from lower values ​​in early diastole (10-20 kPa) and reach much higher values ​​in late diastole (200-500 kPa) indicate that materials used in regeneration should reflect these changes to support proper cell contraction and their integration with the surrounding tissue [110]. With such approaches, cardiac patches have the potential to improve myocardial function and support repair processes after heart attacks or other cardiac injuries [111].

The research conducted by Asadpour et al. [112] concerned the synthesised of biodegradable poly(ester-ether-urethane-urea) (PEEUU) which was blended with polycaprolactone (PCL) -Figure 6- to improve its mechanical and physicochemical properties.

As a result of this process, it was found that the elasticity of the fabricated blends increased with the increasing share of PEEUU. The studies observed that the elastic moduli reached various values: for PEEUU it was 0.62 ± 16 MPa, while for PEEUU-PCL in the proportion of 3/1 it was 12.62 ± 3.1 MPa, and in the proportion of 1/1 it reached 63.3 ± 4.07 MPa. However, the modulus of elasticity of PEEUU films and PEEUU-PCL blends was in the range of values ​​similar to that of native heart tissue, making them promising materials in the context of medical applications. The low stiffness of the resulting materials may be beneficial for integration with native heart tissue, because too stiff polymeric blend may inhibit cardiomyocyte growth and function. Additionally, blending PEEUU with PCL showed good biocompatibility. These blends revealed appropriate cell-scaffold interactions, resulting in increased functional activity of cardiomyoblasts, which could be measured by analyzing the expression of markers specific for heart tissue. These results suggest the potential of using PEEUU-PCL in the context of cardiac tissue regeneration.

Baheiraei et al. [113,114] obtained electroactive polyurethane/siloxane films containing fragments of aniline tetramer. The presence of electroactive aniline tetramer in these materials leads to increased expression of heart-specific genes, which is crucial for cardiac contractile function and electrical signaling. Increased expression of genes such as Cx43 (connectin cadherin), TrpT-2 (receptor protein) and SERCA (calcium pump protein), suggests that these changes may support better intercellular connections and synchronized contractions, which are crucial for efficient cardiac muscle function. Furthermore, these materials were shown to be biocompatible and do not negatively affect the intrinsic electrical properties of HL-1 cells. Incorporation of the electroactive part of polymeric constructs improves their ability to transmit electrical signals between cells.

Electroconductive nanofibrous polyurethane/graphene (PU/G) composites obtained using different concentrations of graphene and two different fabrication techniques (electrospinning and solvent casting) showed interesting electrical and mechanical properties. Research by Bahrami et al. [115] revealed that with increasing graphene content in the materials, their electrical conductivity increased significantly. Graphene acted as electrical bridges, which contributed to the improvement of electrical conductivity of PU/G composites. Additionally, the mechanical properties of composites obtained by electrospinning were significantly better compared to those produced by solvent casting. Biological evaluations confirmed that the introduction of graphene supported cell adhesion and proliferation, suggesting that these composites are non-toxic and can be used in biological and medical applications. The study results indicated that graphene was better distributed in the polyurethane matrix when the electrospinning method was used, which may have a significant impact on the final mechanical and electrical properties of the composites.

In the work of Mani et al., [116] an electrospun new nanocomposite filled with basil oil and titanium dioxide (TiO₂) particles was developed. The composite material showed increased hydrophobicity and reduced fiber diameter compared to the original polymer. The prepared patch delayed the clotting time and provided a safe environment for red blood cells, which indicates its hemocompatibility, and the cellular toxicity of the developed composite was lower than that of the original polymer.

The research conducted by Tao et al. [117] focused on the use of an innovative angiogenic hydrogel based on poly(ethylene glycol) fibrin, reinforced with a biodegradable poly(ether-urethane)urea (BPUR) mesh, for the development of ventricular heart wall replacement in rats. The developed hydrogel scaffold aimed to stimulate cell invasion and the formation of new blood vessels, which is crucial for tissue regeneration. During the research, a significant improvement in regenerative tissue remodeling was observed in places where an artificial defect on the right ventricular wall was introduced. The use of an engineered hydrogel patch reinforced with BPUR contributed to the intensification of blood vessel formation and muscle regeneration, which indicates its potential effectiveness in the treatment of heart damage. Due to the limited fibrosis reaction, a reduced number of arrhythmia cases and improved heart performance were observed in the studied animals. These results suggest that the developed hydrogel scaffold may have significant implications for future therapies aimed at regenerating and improving heart function following heart injury.

The research work of Chen at al [118] presents a biomimetic electrospun anisotropic scaffold based on polyurethane (PU) and ethylcellulose (EC), characterized by uniform fibrous nanostructures and three-dimensional porous networks. The results showed that the anisotropic folds of the scaffolds with aligned nanofibers were successfully fabricated and exhibited high mechanical properties for cardiac cell survival and function.

Immediately after myocardial infarction, there is an overproduction of reactive oxygen species (ROS), which leads to disruption of cellular homeostasis. These disorders contribute to cardiomyocyte damage and promote inflammatory processes and fibrosis in the heart. ROS play a key role in the pathological processes of cardiac remodeling after infarction, associated, for example, with increased activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which is one of the main sources of these reactive species [119].

The study by Yao et al. [119] focused on biodegradable elastomeric ROS-responsive polyurethane containing thioketal bonds (PUTK). This material was synthesized from polycaprolactonediol (PCL-diol), 1,6-hexamethylenediisocyanate (HDI) and a ROS-cleavable chain extender. PUTK was electrospun into fibrous patches that were capable of loading the glucocorticoid methylprednisolone (MP) – Figure 7. These patches were used to treat myocardial infarction (MI) in rats in vivo. In long-term treatment lasting 28 days, the fibrous patches containing MP significantly improved cardiac function and angiogenesis, and effectively reduced fibrosis and negative cardiac remodeling after MI. The results of these studies suggest that PUTK may have great potential to optimize and promote its use in the treatment of myocardial infarction.

One of the major limitations of electrospinning is the difficulty in obtaining large pore sizes in the fabricated structures. Typically used electrospinning techniques focus on creating fibers with very small diameters, which can lead to microscale pore sizes [120].

To overcome these problems, scaffolds have been prepared by thermally induced phase separation (TIPS) technique which allows the control of the scaffold pore size by changing the preparation conditions [121].

Xu et al. [122] described an advanced approach to designing biomedical scaffolds made of polyurethane (PU-PEG-PVCL) that were optimized for mechanical adaptation to the native myocardium. The authors used the TIPS method to synthesize an anisotropic scaffold, which allowed for tuning the mechanical properties of the material by manipulating the polymer concentration and selecting appropriate soft segments, which were: triblock copolymer of poly(ethylene glycol) (PEG), random copolymers of ε-caprolactone (CL) and δ-valerolactone (VL). The aim was to achieve mechanical parameters of the designed scaffold that would resemble those of native myocardium. The authors focused on aspects such as suture retention strength, uniaxial and biaxial mechanical properties, and fracture toughness. The results showed that the optimal scaffold achieved a fracture strength of 20.7 ± 1.5 N, which is comparable to that of native cardiac tissues (20.4 ± 6.0 N). Additionally, the biaxial mechanical stretching of the scaffold was similar to that of the myocardium. Analyzing the possibility of creating innovative biomaterials, the authors combined the optimized scaffold with a hydrogel derived from porcine myocardium, which resulted in the creation of a biohybrid scaffold with a morphology resembling a decellularized myocardium matrix (Figure 8). The SEM images of the obtained biohybrid scaffolds showed the fibrous myocardium ECM distributed on the surface and inside their aligned pores.

In experiments after implantation of such a scaffold in rat models, minimal immune response and improved cell penetration were demonstrated compared to PU scaffolds used alone. The studies conducted underline the advantages of biohybridization of synthetic polymers from biological tissues, which allows for the preservation of favorable tissue properties, such as native extracellular matrix (ECM) components and their biological activity, while obtaining durable and mechanically strong synthetic materials.

5.3.Polyurethane Scaffolds for Cardic Valve

The literature review showed that three polyurethane-based heart valves have been intensively studied over the last decade and have either been clinically tested or are currently being tested. These are: TRISKELE transcatheter valve, SiPUU (LifePolymer) and the first implantation of these PHVs in humans [124] and FGO-PCU (Hastalex®) [125], a unique polymer with incorporated graphene [126]. The TRISKELE transcatheter valve made of POSS-PCU is a trileaflet valve. It consists of a nitinol wire frame, leaflets, a POSS-PCU sealing cuff, and a skirt supporting the sealing cuff. The structural elements of the TRISKELE valve were manufactured separately.

POSS-PCU is a nanocomposite polymer consisting of a hard crystalline segment and soft elastomeric segments in which polyhedral oligomeric silsesquioxane (POSS) nanoparticles are attached as pendant chain functional groups to a poly(carbonate-urea)urethane (PCU) backbone. In vitro studies of this polyurethane have shown increased hemocompatibility and a surface resistant to thrombogenesis [127], biostability [127] and resistance to calcification [128]. The described study focuses on an innovative approach to the production of heart valves, using materials such as POSS-PCU in combination with stainless steel. The production method consisting in immersing the stem in a polyurethane solution allows for obtaining uniform and repeatable hydrodynamic properties (PHV) with a controlled leaflet thickness of 130 ± 10 µm on average. The valve prototypes were tested in different sizes (23, 26, 29 mm) under simulated anatomical conditions, which allowed for checking their functionality in the context of the aorta. The results showed that the TRISKELE valves are characterized by hydrodynamic performance that is at least comparable to the currently used reference models. An additional advantage of the TRISKELE valves is a significant reduction in paravalvular leakage, which is crucial for the safety and effectiveness of implantation. Better anchoring and sealing also indicate the high potential of the TRISKELE valves. The valve is currently in preclinical development [129].

SiPUU is a polyurethane that is synthesized using a modified two-step solution polymerization method, which allows to obtain unique material properties. The soft segments of this polymer are based on α,ω-bis(6-hydroxyethoxypropyl)poly(dimethylsiloxane) and urethane-linked poly(hexamethylene oxide) (PHMO) in a ratio of 80:The hard segments are formed using 4,4'-methylenediphenyl diisocyanate (MDI) and a mixture of 1,3-bis(4-hydroxybutyl)-1,1,3,3-tetramethyldisiloxane (BHTD) and 1,2-ethylenediamine (EDA) in a ratio of 50:In vitro studies have shown that the newly developed polymer is characterized by high oxidation resistance, which is important in the context of its use in a physiological environment. The presence of dimethyl siloxane (PDMS) affects the structure of the polymer, increasing its durability and resistance to environmental factors. Ex vivo thrombogenicity tests have shown that the SiPUU polymer causes virtually no platelet adhesion or clot formation on its surface. Additionally, long-term implantation of the SiPUU valve prototype in sheep did not reveal any differences in thrombogenicity or tissue response compared to the conventionally used biological valve. SiPUU (LifePolymer) valves consist of three main elements: a stent, two or three specially designed leaflets, and a sewing cuff. The leaflets are formed in solution on stents made of polyetheretherketone and then mechanically processed for precise dimensional adjustment. This approach allows for obtaining efficient and functional heart valves that can be widely used in medicine [126].

Foldax®, under the trade name Tria™, recently conducted a first-in-human clinical trial of 15 patients undergoing surgical replacement of the LifePolymer valve [127,128,129,130]. The results of this study suggest that most of the complications observed were not related to the valve chosen or to the surgical procedure itself. However, some results suggested that the occurrence of clots may have been related to the Tria valve suture ring, which is made of polytetrafluoroethylene rather than polyurethane. The polysiloxane-based valve demonstrated good hemodynamic function in this first-in-human experience, which is a positive sign. However, due to the limited size of the population and the need for long-term monitoring, further studies with a larger group of patients are needed to assess the efficacy and safety of this type of valve over a longer period of time.

In [125,131], a detailed evaluation of a nanocomposite consisting of functionalized graphene oxide and poly(carbonate-urea)urethane, known under the brand name "Hastalex"®, was made – Figure 9.

It was compared with GORE-TEX™, a commercial polymer traditionally used in medicine, especially in the context of treating cardiovascular diseases. One of the key factors influencing the high tensile strength of Hastalex is the presence of graphene oxide in the polymer matrix. The high elasticity of this polymer allows the production of thinner leaflets, making it a promising material for applications in heart valves, in particular in transcatheter valve replacements (TVR). Cytotoxicity tests indicate high biocompatibility of FGO-PCU, which is further confirmed by the results of tests on cell adhesion, viability and proliferation. Despite of the promising results, there is currently only one prototype of a heart valve prosthesis made of Hastalex polymer, and there is no data on its hydrodynamic performance and in vivo test results. This indicates the need for further research and analysis before the wide application of this new material in clinical practice [132].

Recently, for the first time Melo et al. [133] developed new environmentally friendly isocyanate-free polyurethanes (NIPU) as potential biomaterials for the production of synthetic heart valve prostheses. Poly(tetrahydrofuran) (PTHF)-NIPU and poly(propylene glycol) (PPG)-NIPU were obtained by mixing bis(cyclic carbonate) polyethers with diamine and triamine. NIPUs are a "green" alternative to traditional polyurethanes, and their low thrombogenic profile makes them promising for use in medicine. When used for the production of valve elements, tested in laboratory conditions, they showed hydraulic properties comparable to those used in clinically recognized bioprostheses, which suggests their high efficiency and safety. Additionally, the resistance to calcification of patches made of PTHF-NIPU is another advantage that may contribute to the longer service life of these implants. This study provides a basis for the future development of isocyanate-free, hemocompatible and hydrodynamically competent heart valve prostheses made from NIPU.

5.4.Polyurethane Scaffolds for Vascular Engineering.

Large-diameter artificial blood vessels with an inner diameter larger than 6 mm are widely used in clinical practice. However, small blood vessels with an inner diameter less than 6 mm often cause implantation failure due to their narrow diameter, easy embolization and low long-term patency rate. Therefore, the development of small (<6 mm) artificial blood vessels for medical applications is of great importance. The high elasticity of PU can withstand repeated stress, mimicking the native blood vessel subjected to blood flow.

The work of Nafiseh Jirofti et al. [138] suggests that optimized co-electro-spun PCL/PU grafts can provide sufficient mechanical stress to conform to and mimic the native blood vessels. Biodegradable elastomeric PU was designed as a drug-eluting stent coating to be non-thrombotic and able to provide antiproliferative drug release to inhibit smooth muscle cell proliferation.

Zhen et al. [139] developed biostable, cross-linked polyurethane formulations which were further used to fabricate scaffolds with precisely designed 40 μm pores. The mechanical properties of these scaffolds were matched to those of native blood vessels by optimizing the polyurethane composition. The studies showed that these scaffolds support the healing process and mitigate the body's foreign body response (FBC). The biomaterial obtained is characterized by tunable mechanical properties and a precisely designed porous structure (Figure 10), making it an promising candidate for applications in pro-healing vascular grafts and in situ vascular engineering.

Mostafavi et al. [140] developed a novel sodium alginate-based supramolecular bioactive elastomer (BASPU) that exhibits high strength and advantageous mechanical properties. BASPU utilizes two different physical networks: the first consists of soft ammonium-soft tertiary sulfate pairs that function as strong ionic bonds, and the second consists of soft tertiary carboxylate groups that function as weak bonds. The presence of sulfate groups in the elastomer structure contributes to a low Young's modulus, which means that the material is flexible while offering high strength and excellent tensile properties. Additionally, BASPU exhibits efficient energy dissipation, ultrafast self-healing ability, and high overall healing efficiency. In vitro studies have shown that BASPU has improved endothelial cell adhesion, which is important for integration with biological tissues. In addition, this material demonstrates higher antithrombotic capacity and significantly lower platelet adhesion compared to commercial artificial vessels.

Kianpour et al. [141] presented a novel approach to the production of scaffolds for artificial vascular grafts using a flexible, porous, and biocompatible titanium dioxide nanotube (TNT) film with polyurethane (PU). The study compared the mechanical and biological properties of open (OE) and closed (CE) TNT-PU films with their pure PU counterparts. The results of the study showed that the structure of the OE-TNT-PU scaffolds was not only hydrophilic but also superhydrophilic, which promoted better cell attachment and proliferation. It was noted that the endothelialization rate in the OE-TNT-PU scaffolds was twice as high as that of pure PU scaffolds after 5 days of in vitro culture. Moreover, these scaffolds were characterized by significantly lower platelet counts and agglomeration, which may indicate better biocompatibility of the material. The mechanical properties of the OE-TNT-PU films were considerably improved, compared to pristine PU: the elongation at break was as high as 881% and the tensile strength was more than 3 times higher than that of pure PU. The Young's modulus of these scaffolds exceeded 150 MPa, suggesting that these materials are exceptionally flexible and strong, which is crucial for applications in regenerative medicine and vascular surgery.

Small-diameter vascular scaffolds developed by co-electrospinning using poly(ε-caprolactone) (PCL) and flexible polyurethane (PU) exhibit promising mechanical and biological properties [138]. In vivo studies conducted in rat and sheep models confirmed that increasing the PU content in the scaffolds significantly improves their biocompatibility, reaching 43% with a maximum PU content of 90%. Ultimate tensile strength (UTS) measurements for different PU proportions (10%, 25%, 50%, 75% and 90%) indicate variability in mechanical properties, with the lowest UTS value obtained for 75% PU (2.2 ± 0.34 MPa) and the highest for 10% PU (4.7 ± 0.34 MPa). Cytotoxicity studies conducted using the MTT assay showed that the number of cells on the scaffolds increased compared to the control group, suggesting a beneficial effect of the materials on cell proliferation from day one to day seven. The fabricated composite structures as vascular scaffolds provided appropriate mechanical and biological properties with the desired clinical performance, and the simple preparation method makes the composite structure a good candidate for the required potentials for use as small-diameter vascular grafts in vivo.

Yu et al. [142] prepared by electrospinning hybrid vascular grafts that combines the properties of synthetic thermoplastic polyurethane (TPU) and natural fibroin. The use of a rotating collector allowed obtaining a unique fibrous structure with a biomimetic architecture, which contributed to increased flexibility of the graft compared to traditional, regularly aligned fibers (Figure 11).

The results showed that the obtained fiber morphologies differed depending on the materials and processing parameters used. The TPU/fibroin hybrid grafts showed mechanical properties comparable to those of natural blood vessels. In addition, contact angle tests showed that TPU is hydrophobic, while fibroin is hydrophilic. The combination of these two materials allowed obtaining grafts with balanced hydrophilicity, which favors cellular activity and interaction with the matrix. Importantly, a significantly higher cell population was observed in the case of the TPU/fibroin hybrid graft compared to the graft made of TPU alone. This indicates that the presence of fibroin significantly promotes cell proliferation.

6.Bone Tissue Engineering

Bone grafting is often needed to accelerate the healing of bone defects. In addition to autografting, a standard procedure involving bone taken from the patient's own body (often from the iliac crest), it is possible to use a biodegradable or bioresorbable synthetic material with similar mechanical properties to bone. In particular, tissue engineering approaches to repair or regenerate bone tissue function include the combined use of osteoconductive biodegradable scaffolds, osteogenic cells, and osteoinductive bioactive factors. Signaling molecules such as growth factors can be incorporated into the scaffold to enhance its osteoconductive properties. In terms of mechanical properties, scaffold materials should optimally match the properties of the tissue to be replaced [143,144].

Bone tissue engineering is a rapidly developing field that focuses on creating new methods for treating bone-related diseases. Unlike traditional approaches such as allografts (grafts from other people), autografts (grafts from own body), and xenografts (grafts from other species), bone tissue engineering seeks to find alternatives that could minimize the risk of rejection, infection, and other complications [108].

The design of biomimetic bone material aims to create artificial bone grafts that are superior to autologous and allogeneic grafts and provide full regeneration of bone defects. Based on the definition of biomimetic bone scaffold, Xu et al. [145] proposed that the design and fabrication process of these scaffolds should be based on four key principles:

(1) Compositional biomimetics: Materials should mimic the natural chemical composition of bone, which can affect their biocompatibility and ability to integrate with surrounding tissues.

(2) Structural biomimetics: The structure of the scaffold should mimic the hierarchical organization of bone, which can improve the mechanical properties and functionality of the grafts.

(3) Mechanical strength biomimetics: Materials should have adequate strength and flexibility to meet the biomechanical demands of the body.

(4) Biomimetic regeneration process: Design should take into account the natural processes of bone healing and regeneration, supporting cell proliferation and new bone formation.

Fulfilling these four principles (Figure 12) aims to create biomimetic bone materials that will be a more effective substitute for autologous bone, thus improving clinical outcomes and quality of life for patients.

Bone tissue engineering field encompasses a wide range of medical procedures, including orthopedic defects, bone tumors, spinal segment stabilization, pseudoarthrosis, and various interventions in maxillofacial, orthopedic, reconstructive, and trauma surgery [108].

Bone is a living, dynamic tissue that is constantly remodeling. Studies have shown that 10–15% of the bone in the body is replaced with new bone each year [136]. Bone matrix is ​​a three-dimensional structure secreted by cells into the extracellular space. It is ​​composed of organic (40%) and inorganic (60%) compounds, and exact composition varies with gender, age, and health status. The main inorganic components of ECM are calcium-poor apatite and trace elements. On the other hand, organic ECM is much more complex, consisting mainly of type I collagen (90%) and non-collagen proteins (10%). It is mainly synthesized by osteoblasts before the mineralization process [146].

The bone ECM dynamically interacts with osteoblasts and osteoclasts, regulating the processes of bone remodeling or regeneration. The bone remodeling process occurs thanks to specialized cells of bone tissue, among which we can distinguish [147]:

- osteoblasts, which are responsible for bone synthesis,

- osteoclasts, which are responsible for bone resorption,

- osteocytes and lining cells, which are permanently present in bone tissue.

Due to their structure, location and function, compact and spongy bone are distinguished. One difference between these two types is their porosity. The apparent density of solid compact bone is about 1.8 g/cm³, while the relative density, i.e. the ratio of the apparent density of the sample to the mass of solid bone, reaches a value of > 0.The mechanical properties of spongy bone, which consists of many trabecular elements with dimensions greater than 1 mm, are called apparent mechanical properties. These properties depend on the characteristics of the bone tissue matrix, the amount of tissue and the spatial organization of the trabeculae. The relative density and architecture of spongy bone are of great importance. Since the relative density and architecture of cancellous bone vary depending on the anatomical site, age, and past or permanently acquired diseases, there are significant differences in bone stiffness and strength [148].

Depending on the extent of the defect, we can distinguish three methods of bone regeneration: (1) synthetic scaffolds, (2) scaffolds combined with active particles, and (3) materials obtained in combination with cells. Tissue engineering allows an alternative approach to the creation of bone grafts by providing temporary, artificial scaffolds for the growth and regeneration of natural tissue [149]. In relation to bone tissue, tissue engineering methods allow the production of a three-dimensional (3D) structure with controlled porosity in order to create an environment supporting bone growth. It is desirable to obtain bone scaffolds from biodegradable materials, which is why they are completely replaced by new tissue [150]. A bone scaffold is a mechanical structure designed to mimic the extracellular matrix (ECM) of bone tissue, which creates conditions for bone remodeling processes to occur with minimal complications [151]. Implantation of bone scaffolds is successful if mesenchymal stem cells (MSCs) contact and adhere to the material surface [152]. After the initial stage, bone begins to grow on the outer surfaces of the scaffold with an internal mineralization gradient [153].

The success of this process depends on several critical parameters of the scaffold, such as surface roughness, internal architecture, porosity, chemical composition of the material and biocompatibility [154]. Bone scaffolds in tissue engineering can be made of various types of materials, such as metals, ceramics and polymers. Metal scaffolds are used due to their osteoinductive properties, but they are not biodegradable. Ceramic scaffolds exhibit biomimetic properties, but are stiff, have limited biodegradability and limited processability. Polymer scaffolds are characterized by good processability, controlled degradation rate, as well as the ability to adjust mechanical properties and wettability to the conditions of their use, which makes them the appropriate candidates in bone tissue regeneration [155]. To achieve the optimal biomimetic effect, it is advisable to combine the benefits of several materials in accordance with biomimetic therapy principles.

6.1. Polyurethane Scaffolds for Bone Engineering

Biodegradable polyurethane elastomers, thanks to their tuneable mechanical properties, are excellent materials for bone-tissue engineering. In order to improve the osteogenicity of biodegradable polyurethane scaffolds, they are combined with an osteogenic material. An example of this type of modification is the work of McEnery et al. [156], who synthesized a new thioketal (TK) diol and then cross-linked it with a lysine triisocyanate (LTI) prepolymer. The obtained poly(thioketal urethanes) (PTKUR) are hydrolytically stable, but are subject to degradation by reactive oxygen species (ROS) released by cells. They were then combined with MasterGraft™ (MG; Medtronic) ceramic blend (85% β-tricalcium phosphate/15% hydroxyapatite) to produce PTKUR/ceramic composite cements that, when implanted into femoral condyle defects in rabbits, supported appositional new bone growth, osteoclast-mediated resorption, and host bone integration observed at 6 and 12 weeks after implantation. The cements also demonstrated initial compressive strengths exceeding that of trabecular bone, and working times comparable to commercial bone cements (5–10 min).

McGough et al. [157] investigated PTKUR/ceramic composites as iliac crest (AG) autograft extenders. PTKUR/AG and PTKUR/CaP/AG showed operating times of 10-20 min and compressive strengths of 1-2 MPa. When implanted in a rabbit posterolateral intertransverse process bone formation model, bone growth along the posterior plane of the processes was consistent from 2 to 8 weeks for PTKUR/AG and PTKUR/CaP/AG. New bone formation was also evident in the intertransverse process (ITP) space away from the processes. Incorporation of PTKUR/CaP/AG showed that the AG content could be reduced to approximately 50% without significantly impairing bone healing. This work provides evidence for the high potential of PTKUR-based AG extensions in the treatment of bone defects.

To enhance the colloidal stability, handling properties, and osteoinduction of nanocomposites, the same research group [158,159] introduced hydroxyapatite nanocrystals (nHA-PTKUR) into PTKUR, which were implanted into femoral defects in white rabbits to assess ossification at 4, 12, and 18 months of age. The results suggest that nHA-PTKUR cements support combined intramembranous and endochondral ossification, which results in enhanced cement osseointegration and potentially may improve patient outcomes.

Talley et al. [160,161] synthesized low-viscosity polyester polyurethane (LV-PUR) and then obtained the composites by doping with 85% β-tricalcium phosphate/15% hydroxyapatite (CM) or bioactive glass (BG). They also incorporated recombinant human bone morphogenetic protein-2 growth factor (rhBMP-2), which promotes the differentiation, maturation, and proliferation of multivascular cells. Injectable bone grafts were placed in a rabbit posterolateral fusion (PLF) model. μCT images and histological sections revealed evidence of bone union. This study highlights the potential of rhBMP-2-loaded LV grafts as injectable bone grafts for MIS posterolateral spinal fusion surgery.

Studies by Lavrador et al. [162] have shown that biodegradable polyurethane (PU) foam with synthesized polycaprolactone (PCL) and HMDI segments, combined with a 1,4:3,6-dianhydro-d-sorbitol chain extender, has potential in medical applications, especially in tissue engineering. Scaffolds made of this foam, enriched with nano-hydroxyapatite (nHA) and bioactive compounds (such as creatine/putrescine or soy lecithin), were implanted into defects in the tibial and iliac crest of sheep. After 26 weeks of observation, new bone growth was observed both around and inside the PU scaffolds. These results emphasize the importance of the porosity of these scaffolds, which allows cell infiltration and promotes bone tissue regeneration processes. This study suggests that appropriately designed biomimetic materials may support bone regeneration and be useful in the treatment of bone defects.

Lei et al. [163] in their study focused on the development of a porous polyurethane adhesive (PUA) and the evaluation of the influence of different components, such as water, polyisocyanate and β-tricalcium phosphate (β-TCP), on the physicochemical and mechanical properties of this material. By modifying the water content, PUA with different surface morphology and porosity was obtained, which had a direct impact on its properties. The addition of polyisocyanate and β-TCP proved effective in increasing both the adhesive strength and mechanical properties of the adhesives. Importantly, the adhesion strength of PUA to bone was twice that of clinical bone cement based on poly(methyl methacrylate), suggesting its potential advantage in medical applications. In vitro analyses, including cell culture and adhesion tests, showed high biocompatibility of PUA. In addition, in vivo studies in a rabbit model showed that the porous structure of the adhesive promotes cell growth and bone tissue regeneration. PUA also showed compressive strength and modulus that were similar to those of cancellous bone, which is an important advantage in the context of its use as a bone gluing material. All these results indicate that the porous polyurethane adhesive has the potential to become an effective solution for bone regeneration and reconstruction through bone gluing.

In the work of Zhu et al. [164] one investigated the ability of TiO₂ nanoparticles to improve the mechanical properties of scaffolds, promote biomineralization and support the proliferation of osteogenic cells. Since unmodified TiO₂ nanoparticles cannot distribute evenly in the fibers and do not show the reinforcing effect, the poly(ester urethane)urea (PEUU) scaffold was first reinforced by grafting TiO₂ onto poly(ester urethane) (PEU) to obtain PEU-g-TiOThe scaffolds were fabricated by electrospinning to obtain three kinds of fibers: pure PEUU scaffold, PEUU scaffold with unmodified TiO₂ nanoparticles (TiO₂/PEUU) and PEU-g-TiO₂/PEUU. PEU-g-TiO₂/PEUU significantly increased the Young's modulus and tensile stress of PEUU scaffolds, while unmodified TiO₂ significantly decreased the Young's modulus and tensile stress. The greatest strengthening effect was observed for the 1:1 ratio PEUU and TiO₂-modified PEU scaffolds. PEU-g-TiO₂/PEUU scaffolds also showed a higher biomineralization capacity than PEUU scaffolds. Moreover, these scaffolds promoted MSC growth better than pure PEUU scaffold and TiO₂/PEUU scaffold. The obtained scaffolds, alone or in combination with osteogenic cells, have the potential to regenerate bone.

Synthesized by Huang et al. [165], a novel levofloxacin loaded mesoporous silica microspheres (MSN)/nano-hydroxyapatite /polyurethane composite (HA/PU) scaffolds represent an innovative approach to the treatment of bone defects (Figure 13). As a result of this material’s composition, a degradable scaffold was formed that not only releases drugs but also exhibits bioactivity, which promotes bone tissue regeneration. The therapeutic effect in the treatment of chronic osteomyelitis with bone defects was evaluated in a rabbit model compared to bulk PMMA. The degradation of this composite starts after 12 weeks from implantation, which ensures that until then the material maintains its structural integrity, offering adequate mechanical support for the bone healing process. After the degradation of the material, new bone tissue is formed. This novel antibiotic-loaded biocomposite scaffold may serve as a drug delivery system for treating bone defects due to chronic osteomyelitis. However, as indicated, the use of antibiotics, including levofloxacin, may lead to the development of bacterial resistance.

The use of drugs/antibiotics alone leads to the increase of pathogen resistance, therefore Huang et al. [166] designed another nano-hydroxyapatite/PU (Ag/n-HA/PU) composite containing 3% silver. Silver ions are known for their antibacterial properties and can effectively combat the bacteria responsible for osteomyelitis and infections associated with bone gaps. Good bone defect repair, reasonable degradation rate of scaffolds and no significant toxicity were observed, indicating the advantages of this new synthetic scaffold as a potential treatment option for chronic osteomyelitis.

In general, antibacterial activity in the biomedical perspective is associated with compounds or elements that locally retard bacterial growth or anihilate bacteria without being generally toxic to the surrounding tissues. Most of the current antibacterial agents available in the medical industry are chemical modification of natural compounds [167]. An example of an innovative approach is the work of Zhang et al., [168] who developed an injectable antibacterial bone cement by combining Enoxacin (EN) with a polyurethane/ nano-hydroxyapatite (PUHA) composite -Figure 14.

Enoxacin, a third-generation fluoroquinolone antibiotic, has a broad spectrum of activity, especially against Gram-positive bacteria [169]. Its minimum inhibitory concentration (MIC) is much lower compared to the used inorganic antibacterial agents, such as silver or gold. [170] For this reason, EN is a suitable choice in the context of biomaterial-associated infections (BAI). The antibacterial cements showed good viscosity and injectability with shear behavior ensuring convenient manipulation. Adding EN drug accelerated the conversion of monomers and increased the cross-linking and mechanical properties, which enhanced the stability of the cured EN-loaded cements, which is beneficial to avoid the inflammatory reaction caused by the residual monomers. All the drug-loaded PUHA cements showed antibacterial properties.

In recent years, the use of 3D printing techniques has increased for developing bone substitutes. 3D printing is a flexible method to manufacture a variety of materials such as polymers, ceramics, metals, and composites with unique geometries and macro/microporous architectures. In bone tissue engineering, 3D printing methods have numerous advantages such as the control of fine features including interconnected porosity, there are no contamination issues related with any second material for the support structures, and it showed the ability of direct printing with both metallic and ceramic materials. [171]. 3D printing ink materials should show degradability, biocompatibility, and processability, and their degradation products must not have side effects on human health [172]. For the first time, Ma et al. [173] developed a series of biodegradable piperazine (PPZ)-based polyurethane-urea (P-PUU) scaffolds with a gradient of PPZ content by pneumatic extrusion 3D printing technology. They demonstrated that the P-PUU ink at 60 wt% had a suitable viscosity for fabricating the scaffolds. The 3D-printed P-PUU scaffolds showed an interconnected porous structure with a macropore size of about 450 μm and a porosity of about 75% - Figure 15.

By adjusting the PPZ content of the P-PUU scaffolds, their mechanical properties could be moderated, and the scaffolds with the highest PPZ content showed the highest compressive modulus (155.9 ± 5.7 MPa) and strength (14.8 ± 1.1 MPa). Furthermore, both in vitro and in vivo biological results suggested that the 3D-printed P-PUU scaffolds possessed excellent biocompatibility and osteoconductivity, which facilitated new bone formation.