Synthesis and use of thermoplastic polymers for tissue engineering purposes

Abstract

Thermoplastic polymers provide a versatile platform to mimic various aspects of physiological extracellular matrix properties such as chemical composition, stiffness, and topography for use in cell and tissue engineering applications. In this review, we provide a brief overview of the most promising thermoplastic polymers, and in particular the thermoplastic polyesters, such as poly(lactic acid), poly(glycolic acid), and polycaprolactone, and the thermoplastic elastomers, such as polyurethanes, polyhydroxyalkanoates, and poly(butyl cyanoacrylate). A particular focus has been made on the synthesis processes, the processability and the biocompatibility. We also discuss how these materials can be applied in tissue engineering, mimicking tissues' structure and function, and stimulate mesenchymal stem cells differentiation and mechanotransduction.

Graphical abstract

Highlights

• •Thermoplastic polymers are characterized by remarkable mechanical properties, which could mimic the properties of the biological tissues.
• •Thermoplastic polymers could be effective as implants that require long retention time and also a great stability.
• •Thermoplastic polymers result interesting tools to effectively stimulate MSCs differentiation through mechanostransduction.

1. Introduction

The rapid evolution of tissue engineering highlighted the need to use different materials to develop specific scaffolds for different tissue engineering applications. The role of the scaffolds is pivotal, since they allow the cell homing, adhesion, differentiation, and proliferation, consequently guiding the new tissue formation. In particular, the scaffolds main aim is to replace the tissues' extracellular matrix (ECM) on an interim basis, interacting with living cells (Asadi et al., 2020; Terzopoulou et al., 2022). For these reasons, it is of fundamental importance that the materials used in tissue engineering are biocompatible, degradable, nontoxic, and with adequate mechanical properties (Terzopoulou et al., 2022; Kalirajan et al., 2021).

Nowadays, the great variety of materials available provides a wide range of properties that determine which types of materials are more suitable for the targeted tissue. Notably, it is well known that polymers, both synthetic and natural, represent a versatile platform for the development of different tissues substitutes, since they possess a wide range of advantageous properties, especially in terms of biocompatibility and processivity (Kim et al., 2012). In fact, polymers could be used to mimic various characteristics of the native ECM, such as micro- and nano-topography (Bianchi et al., 2022; Dolatshahi-Pirouz et al., 2011), mechanical properties (Nemir and West, 2010; Kennedy et al., 2017), and also chemical composition (Zou et al., 2018), resulting in biomimetic scaffolds with an optimal microenvironment for the host cells and tissue. The main aim in the development of tissue engineered constructs is to obtain scaffolds possessing a combination of these properties, leading to the mechanotransduction of cell response, fundamental for the stimulation of a specific stem cells differentiation (Kim et al., 2012; Kennedy et al., 2017).

As a matter of fact, a material results compatible also based on its mechanical properties, which need to be similar to those of the host tissue, especially in terms of elasticity as concerns dynamic and structural tissues, such as tendons and ligaments respectively.

A material can be referred to as elastic when it is characterized by reversible deformation with a high resilience, meaning that it can recover after the application of a force (Chee et al., 2013; Gosline et al., 2002). This type of behavior is fundamental for the correct functioning of the biological tissue, and it also represents a major problem for the research to replicate, especially under variable situations where forces of different extent can be applied.

Despite the efforts that have been put into the development of scaffolds able to mimic the host tissues, nowadays there are few examples of tissue engineered constructs available for clinical use and intended for the repair of muscles and connective tissues (Freed et al., 2009; Bianchi et al., 2021). This is principally due to the mechanical incompatibilities between the scaffolds and the tissue. In fact, although a wide range of materials have been well characterized to correspond with these properties, such as thermoplastic polyesters (i.e. poly(lactic acid), poly(glycolic acid), and polycaprolactone), numerous gaps in terms of mechanical properties are still present, leading to scaffolds failure during the experimental animal studies and the preclinical trials (Terzopoulou et al., 2022; Chee et al., 2013).

Recently, elastomers have gained attention in this research field, since they can support great deformation stresses without breaking, consequently recovering their original shape after the force removal. In particular, the thermoplastic elastomers result as the more adequate to the aim, due to their structure which alternates rigid segments, which provide mechanical strength, and amorphous segments, which provide the flexibility (Coleman et al., 1986).

This review will be focused on the description of thermoplastic polymers, in particular thermoplastic polyesters, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and polycaprolactone (PCL), and thermoplastic elastomers, such as polyurethanes, polyhydroxyalkanoates (PHA), and poly(butyl cyanoacrylate) (PBCA) (Fig. 1). The properties of these materials and their advantages in the field of the tissue engineering of dynamic and structural tissues have been reviewed. Along with this, examples of research involving the development of thermoplastic polymers-based scaffolds have also been reported, considering their advantages and disadvantages.

Fig. 1.

Thermoplastic materials principally used in dynamic and structural tissues engineering.

2. Thermoplastic polyesters

Thermoplastic polyesters are condensation polymers also known as linear or saturated polyesters. They are mainly characterized by the presence into their main chains of ester functional groups (–COO–), which influence the polymer properties, since they are susceptible to hydrolysis, ammonolysis, and ester interchange (Lim, 2017). The popularity of these polymers is due to their remarkable properties, such as the fact that they could be degraded through de-esterification by hydrolysis. More interestingly, the monomeric components of these polymers are removed by natural pathways after degradation, as they can be converted to other metabolites or eliminated, resulting endogenous to human body and biocompatible (Terzopoulou et al., 2022; Seal et al., 2001). In particular, the degradation of the thermoplastic polyesters occurs by hydrolytic random scission of the ester bonds (Fig. 2). PGA is degraded into glycolic acid, an intermediate in the photorespiratory carbon oxidation cycle, which is cleared from the human body through urine or may be converted into glycine, which is further degraded into carbon dioxide and expired via the lungs. On the other hand, PLA is degraded to lactic acid, a human metabolic byproduct, by glycogen breakdown from amino acids and from dicarboxylic acid. Its metabolism in carbonic acid is due to an oxidation process in the tissues, and subsequently it is expired via the lungs. As regards PCL, hydroxycaproic acid was identified as the degradation product. Even if the metabolism and secretion is still uncertain, the most likely elimination route seems to be via urine and the expiration from lungs as carbon dioxide after unidentified metabolic pathways in the body (Pappalardo et al., 2019).

Fig. 2.

PGA, PLA, and PCL degradation pathways.

Thanks to these properties, thermoplastic polyesters have been widely used during the last decades for the development of degradable sutures. They have also been approved by the US Food and Drug Administration (FDA) since they have been demonstrated safe. In fact, they do not provoke an immune response as they are deficient in peptide bonds (Seal et al., 2001).

Thermoplastic polyesters are also characterized by a great versatility due to the new advances in monomer chemistry, which lead to a wide variety of modifications of their crystallization behavior, reaching improvements in mechanical, thermal and optical properties. These modifications can occur during both polymerization and compounding stages, and they can include chain extenders, nucleating agents, and stabilizers, resulting also in the enhancement of the polymers hydrolysis resistance.

As it concerns the gaps in performances, such as their excessive rigidity, most of the polyester can be compounded with fillers with the intent of enhancement of the mechanical and physical properties (Lim, 2017).

Over the past decades, these polymers have represented one of the most investigated groups in the tissue engineering field, being particularly dominated by poly(lactic acid), poly(glycolic acid), and polycaprolactone.

2.1. Poly(lactic acid)

PLA is an aliphatic polyester produced from renewable resources (such as wheat, corn, starch, straw, and cellulose) (Balla et al., 2021), and it was firstly synthesized by Carothers in 1932, while a PLA with higher molecular weight was synthesized by DuPont in 1954 (Lunt, 1998; Hamad et al., 2015). It is a long linear chain formed by repeated units of lactic acid (2–hydroxypropionic acid), which exists in two different isomers that are the mirror images of each other (Fig. 3a): the dextrorotatory form, or L(+), and the levorotatory form, or D(−), with the signs (+) and (−) indicating the direction of plane-polarized light rotation (Jem and Tan, 2020; Chen et al., 2016).

Fig. 3.

(a) Stereoisomers of lactic acid; (b) direct poly-condensation of lactic acid; (c) ring-opening polymerization.

Lactic acid monomers can be converted to PLA polymers using various polymerization processes, including a direct poly-condensation of the monomer (Fig. 3b), a ring-opening polymerization (Fig. 3c), and an azeotropic dehydration condensation. However, it is fundamental to start from highly purified lactic acid to obtain PLA with good qualities and a high yield, since it is greatly affected by lactic acid's impurities (Jem and Tan, 2020; Magazini et al., 2010; Oliveira Campos de França et al., 2022). For this reason, the route most used in the industrial field is the lactide ring-opening polymerization, which is referred to as a two-steps process, since it is characterized by a clear intermediate lactide step. In particular, the first step aims at obtaining a lactide with high optical purity, while the second step consists of the polymerization promoted by organic catalysts, such as zinc oxide, tin oxide, zinc chloride, and tin chloride (Oliveira Campos de França et al., 2022; Singhvi et al., 2019). Typically, L–lactic acid is used for the production of commercial PLA, which is called PLLA, due to the fact that the majority of fermentation processes produce the L–isomer (Jem and Tan, 2020; Chen et al., 2016).

PLA is a versatile and strong biocompatible and biodegradable polymer, which nowadays plays an effective role in the medical field. Its use is principally due to the fact that it provides excellent properties at low costs if compared to other biodegradable polymers (Singhvi et al., 2019). In particular, it has gained great attention due to its remarkable mechanical properties, since it is characterized by a Young's modulus of 3–4 GPa, a tensile strength of 50–70 MPa, and an elongation at break of 2–10 % (Chen et al., 2016; Perego and Cella, 2010). Moreover, it is easily processable with various techniques, such as blow molding, extrusion, injection molding, and electrospinning (Singhvi et al., 2019).

However, PLA also possesses disadvantages, such as a poor thermal stability, a high hydrophobicity, and a slow degradation rate, which could limit its applications. In order to overcome these problems, several methods to modify PLA have been studied, such as the use of plasticizers (i.e. glycerol (Perego and Cella, 2010), citrates (Wang et al., 2009), fatty acids esters (Lebarbé et al., 2015), or blending the PLA with other polymers (i.e. polycaprolactone (Chee et al., 2013), and polyethylene glycol (Chieng et al., 2013)).

The main issue that needs to be overcome is PLA degradation rate in physiological environment, which is too slow compared to the tissues regeneration rates. However, this can be tailored to match tissue growth according to the final application, and to differences in pH and temperature (Terzopoulou et al., 2022). The wide range of possibilities to vary PLA degradation time represent the major attracting factor for its use in tissue engineering. In fact, PLA can be used for the production of sutures that need to be degraded fast, and also to develop medical devices to be used for a prolonged time (Da Silva et al., 2018). Recently, various methods have been studied to increase the PLA degradation rate, such as blending, copolymerization, compounding and surface modification. In Table 1 the advantages and disadvantages of each methos are summarized, together with the degradation mechanisms that are involved.

Table 1.

Summary of PLA degradation accelerating methods and their advantages and disadvantages.

Methods	Materials	Degradation mechanism	Advantages	Disadvantages	References
Blending	PEG, PLCL-PEG, PGA, PBS	Surface and bulk erosion; Autocatalytic.	Easy to adopt and operate; Tunable hydrophilicity.	Decrease of the mechanical properties.	Wang et al. (2017) Oosterbeek et al. (2019) Shuai et al. (2021) Hu et al. (2018)
Copolymerization	LLA-GA, PCL, DXO, PULL	Random chain scission; Surface and bulk erosion.	Tunable degradation rate.	Process difficult to control; Changes in the PLA structure.	Pattaro et al. (2020) Qian et al. (2000) Puthumana et al. (2020)
Compounding	g-MgO-WS/PLA, PLA/WF/PMMA, PLA/starch	Surface and bulk erosion.	PLA enriched with other properties, such as improved cell adhesion, proliferation, and osteogenic differentiation.	Weak interface bonds between the components.	Wen et al. (2017) Wan and Zhang (2018)
Surface modification	Alkaline surface hydrolysis, atom transfer polymerization, photografting by UV light, plasma treatment	Autocatalytic; Surface erosion; Chain end scission.	Good hydrophilicity and increased cell adhesion.	Complex process.	Tham et al. (2014) Hazal Baran and Yildirim Erbil (2019)

PEG: poly(ethylene glycol); PLCL-PEG: polyethylene glycol functionalised poly(l-lactide-co-ε-caprolactone); PGA: poly(glycolic acid); PBS: poly(butylene succinate); LLA: l-lactide; GA: glycolide; DXO: 1, 5-dioxepan-2-one; PULL: pullulan; g-MgO-WS/PLA: surface-grafted magnesia whiskers/poly(l-lactide); PLA/WF/PMMA: Polylactic acid/Wood flour/polymethyl methacrylate.

In the human body, PLA mainly undergoes a hydrolytic degradation process, but it was found that also enzymes, such as acid phosphatase and lactate dehydrogenase, are able to increase its degradation rate (Puthumana et al., 2020). This is greatly influenced by both the environment pH and temperature and PLA characteristics, such as crystallinity and molecular weight.

The degradation usually consists of two stages: in the first stage, the ester bond is broken, and PLA is divided into smaller chains by the water molecules, which penetrates the matrix. In this stage the molecular weight decreases producing water-soluble oligomers. In the second stage, biological enzymes catalyze the oligomers' hydrolysis into monomeric lactic acids, which are subsequently further broken down into carbon dioxide and water, to be finally expelled from the body (Feng et al., 2021).

The main reaction at the beginning of PLA degradation is autocatalytic hydrolysis (Limsukon et al., 2019). This means that PLA hydrolysis is catalyzed by the water-soluble oligomers and carboxylic acid chain ends of the monomers produced by PLA degradation. PLA is a semi-crystalline polymer constituted by a crystal region, formed by ordered chains, and by an amorphous region, formed by random chains (Liu et al., 2018). Because the molecular chain is loosely packed in the amorphous region, it is easier for the water molecules to penetrate and start the hydrolysis process. At the breakage of the ester bond, PLA crystallinity increases together with the molecular weight decrease, and the formation of smaller molecular chains causes the gradual degradation of the crystal region and the loss of the polymer cohesive strength. At this point, the PLA chain becomes more hydrophilic and dissolves in the aqueous environment (Feng et al., 2021).

A wide range of devices has been prepared from PLA, since it has been demonstrated to be a suitable bioabsorbable polymer. In particular, fixation devices such as resorbable plates and screws, extensively used by orthopaedic surgeons due to their advantages when compared to metallic plates, were developed, together with drug releasing micro- and nano-particles, and porous scaffolds for tissue engineering applications (Hamad et al., 2015; Singhvi et al., 2019; Lasprilla et al., 2012).

Due to its safety, biodegradability and biocompatibility, PLA has been approved by the US FDA and other regulatory agencies to be implanted in the human body (Suzuki and Ikada, 2010). Various medical implants have been developed, such as stents, bone fixation devices, sutures, and plates, and numerous scaffolds for tissue engineering.

2.1.1. Tissue engineering applications

Tissue engineering is a multidisciplinary approach which aims to induce repair and replacement of tissues which are defective or affected by pathological conditions, by developing biological substitutes (Bianchi et al., 2021). An ideal scaffold should promote the cell adhesion and proliferation, and the production of ECM, while allowing the nutrients and growth factors transport. Moreover, it is of fundamental importance that the scaffold biodegrades with a tunable rate, which should correspond to the one needed for the new tissue formation, without provoking toxicity or induce an inflammatory response (Bianchi et al., 2021; Langer and Tirrell, 2004).

Despite its hydrophobic nature, PLA is one of the most popular thermoplastic polymers investigated in tissue engineering since it is able to preserve its structural integrity while supplying specific cell-surface receptors during the regeneration process (Chen et al., 2016). However, hydrophobic surfaces limit the cell adhesion and proliferation due to the small contact between the surface and the cells, reducing proteins adsorption and resulting in a slowdown of the regeneration process (Ferrari et al., 2019; Tserepi et al., 2016).

To overcome the limitations related to the PLA hydrophobicity, numerous studies have been conducted to combine PLA with bioactive materials. For example, Milanesi et al. fabricated PLA based nanofibers loaded with essential oils by means of electrospinning for wound healing. In order to create a hydrophilic layer onto the fibers surface able to enhance cell adhesion and promote tissue repair, chitosan was chosen as coating agent, since it represents one of the most attractive biomaterials for wound healing applications. The chitosan coating led to an increase in the surface hydrophilicity, consequently promoting cell adhesion and proliferation and resulting in scaffolds completely colonized by cells (Milanesi et al., 2021). Similarly, in another work a 3D printed scaffold based on PLA was developed and coated with chitosan in order to combine PLA mechanical strength and chitosan bioactivity for bone regeneration. It was found that the coated scaffolds maintained remarkable mechanical properties. Moreover, they could also increase mesenchymal stem cells adhesion and osteogenic activity, together with an enhancement of the scaffolds angiogenic potential (Salehi et al., 2023). 3D freeze-dried porous scaffolds based on PLA combined with chitosan and collagen were also developed for cartilage tissue engineering applications. PLA was combined with collagen and chitosan alone or in association, in order to mimic the natural cartilage tissue environment of chondrocytes. While PLA gave a mechanically stable skeleton to the scaffolds, the hydrophilic components, in particular the collagen, led to an increase in the chondrocytes adhesion and penetration into their porous structure (Haaparanta et al., 2014).

The application of PLA-based scaffolds has been widely studied especially in the bone tissue engineering field. In fact, such scaffolds require higher mechanical properties than other tissues in order to mimic the native bone (Bose et al., 2012). At this purpose, Grémare et al. developed 3D printed scaffolds with different pore dimensions in order to investigate the influence of the pore size on the mechanical properties. They could evaluate that the modification of pore size was insufficient to induce a significant difference of the mechanical properties of the scaffolds, which maintained a sufficient integrity to allow easy handling and cell growth (Grémare et al., 2017). Also, Gomes et al. developed starch-PLA fibrous scaffolds, which were characterized by adequate mechanical properties and porosity, as they were also able to support cell adhesion and proliferation and tissue ingrowth upon implantation. Moreover, they found that the scaffolds were also susceptible to enzymatic degradation, resulting in a significant increase in porosity and available space for cell and tissue ingrowth (Gomes et al., 2008).

In summary, PLA-based scaffolds have been successfully tested on a wide range of tissues, such as bone, skin, bladder (Engelhardt et al., 2011), vascular (Abdullah et al., 2019; Weijie et al., 2016), liver (Lv et al., 2007). In particular, its great versatility is due to the fact that numerous fabrication methods could be used to develop PLA scaffolds, including electrospinning (Polonio-Alcalà et al., 2019), 3D printing (Dussault et al., 2022), gas foaming (Shah Mohammadi et al., 2014), and solvent-casting (Kang et al., 2022), which could lead to a great variety of shapes and morphologies.

While its use in tissue engineering presents several advantages, such as biocompatibility, biodegradability, and versatility in scaffold design, making it a popular choice for various tissue engineering applications, it also has limitations that must be addressed for optimal performance. In fact, PLA's biological limitations necessitate ongoing research and modifications to enhance its performance and applicability in clinical settings.

2.2. Poly(glycolic acid)

Similarly to PLA, PGA is a biodegradable thermoplastic polymer characterized by good mechanical properties and greatly studied nowadays in biomedical applications due to its biocompatibility. Firstly synthetized in 1940s, it has been used in oil and gas industry and for packaging development, and it was also the first synthetic absorbable suture (Jem and Tan, 2020; Chu, 2013).

PGA is formed by units of glycolic acid (Fig. 4a), which is the smallest alphahydroxy acid containing both carboxyl and alcohol groups, and it can also be obtained from both petroleum and natural sources, such as pineapple, sugarcane, and sugar beets (Jem and Tan, 2020; Lapporte and Toland, 1972).

Fig. 4.

(a) Glycolic acid; (b) poly-condensation of glycolic acid; (c) ring-opening polymerization.

As PLA, PGA can be synthetized by two polymerization methods: the poly-condensation of glycolic acid (Fig. 4b) and the ring-opening of glycolide (Fig. 4c) (Ayyoob et al., 2017; Niaounakis, 2015; Ashgari et al., 2017). Although poly-condensation is a straightforward procedure, it is not very effective in producing high molecular weight PGA. This is due to water being produced as a byproduct of the PGA condensation polymerization, resulting in the restriction of the finished product molecular weight. Moreover, PGA possesses low thermal stability above its melting point and fort his reason the poly-condensation reaction could quickly deteriorate it, since its temperature is quite close to PGA melting point (Ayyoob et al., 2017).

For this reason, ring-opening polymerization is the most used in order to obtain PGA with high molecular weight, processability, and mechanical strength with the less residual monomer (about 1–3 %) (Jem and Tan, 2020; Niaounakis, 2015).

PGA possesses a good solvent resistance to the most common organic solvents, as it is only soluble in hexafluoroisopropanol (Gautier et al., 2009). Moreover, it is characterized by a melting temperature of 220–230 °C, and a heat distortion temperature of about 170 °C, which could result advantageous for its sterilization process and high temperature uses (Gautier et al., 2009; Singh and Tiwari, 2010).

PGA is characterized by higher mechanical properties than other biodegradable polymers and plastics, probably due to its crystalline structure that is reported to have a planar zigzag conformation (Lee et al., 2017). PGA possesses a tensile strength of about 115 MPa and a Young's modulus of about 7 GPa (Jem and Tan, 2020). Moreover, PGA possesses barrier properties to gas molecules with low permeability due to its high stereochemistry structure, which render it an optimal material for packaging applications (Jem and Tan, 2020; Magazini et al., 2010).

Interestingly, PGA is also characterized by good hydrolytic degradation property, due to its extremely hydrolysable backbones. In particular, it has been studied that the degradation of PGA in water consist of two main stages: the first stage involves the degradation in the amorphous region with a consequent increase in crystallinity, while the second stage involves the degradation in the crystalline region (Hakkarainen et al., 1996; Magazzini et al., 2021). Moreover, similarly to PLA, PGA degradation starts with a process of autocatalytic hydrolysis. However, the PGA degradation time is shorter than PLA (1.5–3 months in vitro) considering comparable molecular weight (MW) (Zhang et al., 2020). Moreover, the combination between PLA and PGA can lead to a product, the PLGA, with tunable degradation properties based on the PLA:PGA ratio. For example, 50:50 PLGA degrades in 1–2 months, while 75:25 PLGA, and 85:15 PLGA degrade in 4–5 months, and 5–6 months respectively, rendering it an interesting alternative to traditional plastics and a possible enhancer of the biomedical products degradation rate (Terzopoulou et al., 2022). However, the scaling-up of PGA, in contrast with PLA, have not been adequately researched since the monomer glycolic acid results more expensive than lactic acid (Jem and Tan, 2020).

2.2.1. Tissue engineering applications

Together with PLA, PGA is one of the most popular thermoplastic polymers in tissue engineering especially thanks to its biodegradable structure. In fact, while PLA requires 12–18 months to be degraded, PGA degradation occurs after 1.5–3 months considering similar MW, resulting adequate for short-term tissue engineering scaffolds (Khajavi et al., 2015; Budak et al., 2020).

PGA-based tissue engineered scaffolds have been widely investigated for numerous medical applications. In particular, many studies led to the development of nanofibers based on PGA and combined with bioactive materials to increase the host cells adhesion. As a matter of fact, Kobayashi and co-workers produced nanofibers composed of PGA and collagen to induce the scaffolds immediate cellularization and the peripheral blood vessels recruitment. During in vivo tests, they were able to observe that the nanofibers were completely populated and vascularized 5 days after the implant. Moreover, the scaffolds accelerated the inflammation reaction, consequently stimulating the fibroblasts and endothelial cells recruitment from the host tissue (Kobayashi et al., 2013). In another work, a collagen(Col)/PGA/hydroxyapatite(HA) composite scaffold was developed using a 3D printing technique for osteochondral regeneration. The scaffold proved to be a promising tool for cartilage and bone repair, since it was characterized by adequate degradation and mechanical properties. Moreover, when immersed in a simulated body fluid, it led to the upregulation of osteochondrogenesis, showing specific differentiation for both bone and cartilage ECM (Nguyen et al., 2022).

Also, a scaffold based on PGA and silk fibroin was developed for bone defects treatment. The scaffold possessed high mechanical properties and stiffness, and a slow degradation rate in 12 weeks, which corresponds to the physiological requirements for the treatment of bone defects. Moreover, the scaffold enhanced cell adhesion and proliferation, and increased mineralization and bone formation in vivo in 8 weeks (Yao et al., 2022). One other possible application involved the development of a PGA porous three-dimensional matrix as tissue-engineered vascular graft. The system was easily sutured to native blood vessels and it improved the delivery of nutrients and oxygen to the endothelial cells. Moreover, it was estimated its compete degradation after 15 weeks, which corresponds to the stabilization of the vascular blood vessel (Lee et al., 2023).

In summary, PGA have been successfully used to develop scaffolds for a wide range of applications, reaching important results in terms of tissue repair and resulting suitable for the regeneration of blood vessels and cartilage, but also tooth (Ohara et al., 2010), bone (Wang et al., 2010; Dunne et al., 2010), tendon (Xu et al., 2010), and intestinal (Liu et al., 2016).

PGA is recognized for its biocompatibility, biodegradability, and ability to support cell growth, making it a suitable scaffold material. Moreover, it allows a versatile scaffold design, since it could be engineered into various structures, enhancing mass transfer and mechanical strength, which are essential for the development of tissue constructs. However, it also has limitations that must be considered, such as costs and sourcing. In fact, the production of PGA could be more expensive compared to natural biomaterials, which may limit its accessibility for widespread use.

2.3. Polycaprolactone

Together with PLA and PGA, PCL is the thermoplastic synthetic polymer most frequently used in tissue engineering. It was firstly synthesized by Natta et al. in the early 1930s from ε-caprolactone (Fig. 5a) (Chen et al., 2013, Chen et al., 2015). It is mainly studied for its potential application as wound dressing material due to its excellent physicochemical properties. In fact, it is a semicrystalline aliphatic linear polyester composed by repeated units of hexanoate and characterized by high mechanical properties, hydrophobicity, and a slow degradation rate (from several months to several depending on the molecular weight and the degradation conditions) (Chen et al., 2015; Woodruff and Hutmacher, 2010; Labet and Thielemans, 2009; Guarino et al., 2017).

Fig. 5.

(a) ε-caprolactone; (b) ring-opening polymerization.

The conventional synthesis of PCL can occur with two methods: the condensation of 6-hydroxycaproic (6-hydroxyhexanoic) acid, and the ring-opening polymerization of the ε-caprolactone (Fig. 5b) (Chen et al., 2015; Labet and Thielemans, 2009). However, a few papers detailly describe the polycondensation process to obtain PCL, since the ring-opening polymerization is the preferred route, leading to PCL with higher molecular weight and lower polydispersity (Labet and Thielemans, 2009).

PCL has been widely studied in the biomedical fields, and especially as drug delivery system and for the development of scaffolds in tissue engineering, due to its biocompatibility, biodegradability, and also its tunable properties. In fact, it can be easily combined with other polymers in order to create more degradable and biocompatible platforms (Abrisham et al., 2020).

More importantly, PCL has already the FDA approval, and it has been widely used for the fabrication of medical products already launched in the market, such as sutures and bone screws (Chen et al., 2015). However, the deficiency of functional groups in PCL, and properties such as hydrophobicity have limited its application in tissue engineering, since they can lead to poor cell adhesion and uncontrolled biological interactions (Chen et al., 2015; Place et al., 2009). In fact, it is necessary that the surface of a tissue engineered scaffold possesses adequate hydrophilicity, binding sites, and functional groups to avoid the host's foreign body reaction and enhance the cell anchorage, proliferation, and differentiation processes.

To overcome this problem, the introduction of functional groups into the PCL structure has been investigated, leading to the possibility of tuning PCL physicochemical properties, improving its hydrophilicity and also its degradation rate. This renders PCL more suitable for a wide range of biomedical applications (Chen et al., 2015; Xiao et al., 2014; Seyednejad et al., 2011). Moreover, modifications can enhance its properties for specific applications. Various studies have demonstrated that altering the chemical structure of PCL can lead to improved performance in biomedical contexts.

The introduction of functional groups into PCL structures is achieved through various innovative strategies, including ring-opening polymerization, copolymerization, multicomponent polymerization, and oxygen plasma treatment, each facilitating the incorporation of diverse functional groups.

Ring-Opening Polymerization is a versatile method to produce tailored materials with specific properties. This process can be initiated through various mechanisms, including anionic, cationic, and radical pathways, allowing control over polymer architecture, molecular weight, and end-group functionality (Trzebicka et al., 2024). Du et al. used the ring-opening polymerization in combination with click chemistry to create PCL with sulfobetaine and N,N-dimethylamine groups, resulting in systems suitable for drug delivery with enhanced stability and biocompatibility (Du et al., 2024).

An innovative approach is represented by the One-Pot Chain Polymerization and Polycondensation, which combines two polymerization techniques in a single reaction vessel, enhancing efficiency and reducing waste. This method allows for the sequential formation of PCL with specific functional properties. It begins with chain polymerization, often initiated by bifunctional catalysts or initiators. For instance, hydroxymethylphosphonic acid is used to initiate PCL polymerization, introducing phosphoacidic groups along the polymer chain. The initial polymerization is followed by polycondensation, where reactive end groups from the polymer chains react to form longer chains, significantly increasing the molecular weight (Kaluzynski et al., 2022). However, challenges still remain in controlling the reaction conditions to achieve desired polymer properties consistently.

Another novel approach is the Selenide-Mediated Radical Polymerization, which utilizes selenium-containing compounds as mediators to control the polymerization process. The resulting PCL can possess vinyl-terminated chain ends, enabling further functionalization for applications in biomedicine. For instance, the use of ε-(α-phenylseleno) caprolactone allows for the introduction of carbon–carbon double bonds and various side groups through oxidative elimination and thiol–ene reactions, creating multifunctional PCL (Wu et al., 2019). Anyhow, while selenide-mediated radical polymerization offers significant advantages in terms of control and functionality, it is essential to consider the potential challenges, such as the stability of selenium compounds and their environmental impact.

Oxygen plasma treatment is a common, rapid and efficient method, which allows for quick processing times and adaptability. It was used to modify fibrous PCL scaffolds in order to decrease their hydrophobicity, consequently enhancing cell adhesion. This technique effectively introduces a variety of oxygen-containing functional groups (such as –OH, and C O), significantly improving the surface properties, such as wettability, without altering their bulk characteristics (Abdeltwab and Atta, 2022). The process leads to improved wettability, as evidenced by a significant decrease in water contact angles, allowing also a precise control over surface nanotopography, which can be tailored for specific applications, enhancing biocompatibility in medical devices (Chytrosz-Wrobel et al., 2023).

Similarly, the introduction of hydrophilic functional groups to PCL chains using potassium permanganate was used to modify membranes for tissue regeneration. This modification led to enhanced water uptake and biofunctionality while maintaining mechanical strength, indicating improved biocompatibility (Shen et al., 2023).

In alternative, the incorporation of materials like tea tree essential oil (TTEO) and zinc oxide nanoparticles (ZnO-NPs) into PCL-PLA composites resulted in membranes that showed good biocompatibility and smooth surfaces, promoting cell attachment (Grande-Tovar et al., 2023).

Surface modifications are currently used also to improve PCL scaffolds biodegradability. The introduction of hydrophilic groups through copolymerization with hyperbranched polyglycidol (HBPG) led to PCL gels that demonstrated enzyme-triggered disassembly, enhancing biodegradability (González-Chomón et al., 2023).

However, while these modifications enhance PCL's suitability for tissue engineering, it is essential to consider that excessive alterations may compromise its mechanical integrity or lead to unpredictable biological responses. The balance of these factors is crucial for its successful applications in regenerative medicine. Moreover, challenges remain in achieving uniform distribution and stability of the introduced groups, which can affect the material's performance in practical applications.

2.3.1. Tissue engineering applications

As already mentioned, the surface and topography of the scaffold, together with the mechanical properties, should be compatible with the surrounding tissues, in order to provide proper binding sites for the cells and the adequate diffusion of nutrients (Bhullar et al., 2017). At this purpose, Nazeer et al. combined the mechanical properties of PCL with the bioactivity of silk fibroin, a natural protein, in order to fabricate nanofibrous scaffolds by means of electrospinning. PCL was synthesized by means of ring-opening polymerization using an organic diamine as initiator. The incorporation of silk fibroin into the polymeric structure led to a significant change in the surface wettability. In fact, it changed from hydrophobic, with a contact angle of about 130°, to superhydrophilic, with a contact angle of 0°. Moreover, the scaffolds maintained good mechanical properties, almost comparable to that of human skin, and the loading of silk fibroin led to a significant improvement in cell proliferation compared with pure PCL, resulting as promising substrates for tissue engineering applications (Nazeer et al., 2019).

As regards bone tissue engineering, silver nanoparticles were encapsulated into PCL matrix, obtained by means of 3D printing, in order to obtain scaffolds with adequate mechanical properties and also antimicrobial properties that could reduce infections at the site of implantation. The scaffolds were characterized by an interconnected porous structure with mechanical properties suitable for the targeted tissue (1 GPa of Young's modulus). Moreover, the scaffolds well supported osteoblasts proliferation and inhibited the growth of Escherichia coli (Fig. 6), presumably due to the silver release from the polymeric structure (Radhakrishnan et al., 2021).

Fig. 6.

Soft agar plates with Escherichia coli soft agar plates incubated with PCL and PCL‑silver (1Ag2, 3Ag2) scaffolds for 24 h (scale bar: 1 cm). Adapted with permission from (Radhakrishnan et al., 2021). Copyright 2023 Elsevier B.V.

Various studies have been conducted to improve the PCL surface wettability and consequently cell attachment and proliferation. For example, Ehtesabi and Massah (2021) used biocompatible and stable carbon dots for the physico-chemical modification of PCL scaffolds obtained by means of electrospinning. The results showed that the carbon dots addition not only improved the surface wettability, with contact angle change from 84° for the pristine PCL to 0°, but also the mechanical properties, and in particular the systems elongation at break. Moreover, the increase of the surface wettability led to an improvement of the mesenchymal cells adhesion and proliferation compared to the scaffolds of PCL alone (Ehtesabi and Massah, 2021). Similarly, a fibrous structure with nano-nets based on PCL and combined polyethylene glycol was developed. The combination of polyethylene glycol and nano-nets increased the scaffolds hydrophilicity together with the mechanical properties. Moreover, the composite scaffolds were characterized by a large increase in cell density after 3 days of culture, in association with the improved wettability, and more interestingly by an enhanced mineralization with the formation of an apatite layer after 1 week (Tiwari et al., 2017).

In summary, PCL structure could be modified to enhance its application in various fields, resulting suitable for the regeneration of bone (Siddiqui et al., 2021), skin, corneal tissue (Salehi et al., 2021), heart valves and vessels (Janmohammadi and Nourbakhsh, 2019), and nerves (Hu et al., 2016).

PCL's unique properties make it a versatile scaffold material, offering significant benefits for tissue engineering, including biocompatibility and mechanical strength. However, certain limitations must also be considered, such as hydrophobicity and slow degradation, which necessitate careful consideration and potential modifications to achieve an optimal application.

3. Thermoplastic elastomers

Degradable thermoplastic elastomers only emerged in the late 1990s in the field of tissue engineering for the development of FDA Class III medical devices (Chen et al., 2013). They have been used alone or as part of a more complex system for a wide range of diagnostic and therapeutic procedures thanks to their great processability and also to their mechanical and physico-chemical properties. They typically consist of macromolecular long-chains characterized by a reversible mobility when deformed above their softening temperature (Basak, 2021). At room temperature, depending on the microstructure topology, crystallinity, and morphology, the thermoplastic elastomers act as viscoelastic solids. While above their softening temperature the inter- and intra-molecular forces are dominated by the thermal movements caused by the chain segments, resulting in a significant deformation. Although these polymers act as conventional plastics, they are characterized by an inner capacity to be treated and molded in different shapes, ability that render them an important class of polymers to develop biomedical devices (Basak, 2021; Datta and Kasprzyk, 2018). In fact, thermoplastic elastomers can be subjected to a wide range of deformation degrees without breaking and as previously reported, return to the original shape once the load is removed.

Based on the structure linking the chains together and imparting the elastic response, thermoplastic elastomers can be classified as physically crosslinked. This means that their flexible chains could be held together by crystalline regions, glassy domains, weak hydrogen bonds, or dipolar forces (such as the rigid segments of polyurethanes) (Chen et al., 2013). In particular, the rigid segments act as crosslinking agents, providing the mechanical strength and preventing close chains slipping from each other when subjected to deformation, while the soft segments give elasticity (Coleman et al., 1986).

In this section, thermoplastic polyurethane, polyhydroxyalkanoates, and poly(butyl cyanoacrylate) will be described together to their applications in tissue engineering.

3.1. Thermoplastic polyurethane (TPU)

Medical grade TPU has been used in implantable medical devices for decades, such as blood bags, vascular catheters, bladders of the left ventricle assist device, and small caliber grafts for vascular access and bypass surgery (Zdrahala and Zdrahala, 1999). However, recently there is an increasing interest in its application in tissue engineering, due to the fact that it is easily processable, it possesses a wide range of mechanical properties, and it is also characterized by good blood compatibility and resistance to microorganisms' colonization and infections (Bergmeister et al., 2013; Chen et al., 1998). Moreover, polyurethanes chemical compositions, mechanical properties, tissue-specific biocompatibility and biodegradability can be easily modified during the synthesis, gaining a major role in the development of scaffolds in tissue engineering (Chen et al., 2013; Santerre et al., 2005).

TPU structure (Fig. 7a) is composed of aliphatic or aromatic units R1 (usually an aliphatic, aromatic, or alicyclic moiety in the isocyanate monomers) and R2 (usually a more complex group, such as polyether or polyester) linked with polar urethane groups (−NHCOO−) (Kròl, 2007). TPU synthesis involves a urethane-forming reaction between an isocyanate (usually diisocyanate, O=C=N–R1–N=C=O), which forms the chain hard segments, and the hydroxyl group –OH of a bi- or multi-functional polyol (the chain soft segments) in the copolymerization step of diisocyanate and polyols. The use of diisocyanate and a bi-functional polyol results in a thermoplastic polyurethane. Another important component in TPU synthesis is the chain extender, which is used to produce rigid segments, known as extended sequences, in the chain (Fig. 7b). The properties of the TPU are strictly related to the chemical nature and the proportions of these components used during synthesis (Castonguay et al., 2001; Cui et al., 2021).

Fig. 7.

(a) Thermoplastic polyurethane structure; (b) chain extension reaction between a diol chain extender and isocyanate forming urethane.

As already mentioned, TPU is a biocompatible polymer that has been studied for a wide range of applications, from durable devices to biodegradable implants for tissue engineering. The degradation is dominated by the soft segments of TPU's structure (the diol); when their proportion is higher the degradation rate increases (Santerre et al., 2005; Cui et al., 2021).

The degradation could be due to different mechanisms, especially hydrolysis, which affects the aliphatic ester bonds, and enzymatic degradation, such as hydrolytic enzyme action on hydrogen-bonded sites in TPU (Santerre et al., 2005; Santerre et al., 1995). TPUs utilized for the development of scaffolds in tissue engineering are designed to undergo hydrolytic degradation in vivo with the release of non-toxic components, by means of the incorporation of a polyester macrodiol soft segment, such as PLA, PGA or PCL, able to hydrolyse in vivo (Chen et al., 2013; Santerre et al., 1995; Xu and Hong, 2022; Mi et al., 2017).

Additionally, as with the degradation rate, TPUs mechanical properties can also be optimized by changing the ratio between soft and hard segments, reaching values of Young's modulus from 11 to 1690 MPa, elongation at break from 50 % to 570 %, and ultimate tensile strength from 2 to 60 MPa (Zdrahala et al., 1979).

3.1.1. Tissue engineering applications

As already mentioned, TPU's wide range of mechanical properties and biocompatibility led to an increase in its application in tissue engineering. For example, recently it has been widely investigated for the reconstruction of tracheal lesions, since they require materials able to degrade gradually, biocompatible, and with stable mechanical properties. At this purpose, Samat et al. developed a 3D printed tracheal scaffold combining TPU and PLA to increase the properties of both materials. The results showed that a mixture of the polymers led to the formation of scaffolds with the desired Young's modulus (12.2–20.5 MPa), ultimate tensile strength (1–15 MPa), and elasticity (5–10 %). Moreover, higher TPU content led to a gradual degradation in 7 months, suggesting that the scaffold was stable long-term, and an in vitro evaluation demonstrated a cell viability higher than 80 %, proving the systems biocompatibility (Samat et al., 2021). Similarly, in another work TPU has been combined with poly(glycerol sebacate), a non-cytotoxic biodegradable elastomer, to produce vocal fold scaffolds with improved hydrophilicity by means of electrospinning. The addition of poly(glycerol sebacate) led to a decrease in the scaffolds contact angle, and consequently to an increase of their hydrophilicity. Moreover, the scaffolds were characterized by mechanical properties similar to those of the vocal fold lamina ECM. Finally, cells were able to adhere and proliferate onto the scaffolds, suggesting their potential application in vocal tissue engineering (Jiang et al., 2019).

Another possible application of TPU is in the vascular field, in order to substitute autograft vessels and avoid problems related with synthetic grafts, such as thrombosis and hyperplasia. Yu et al. developed TPU scaffolds combined with silk fibroin characterized by an aligned fibrous structure in the inner part and a random fibrous structure in the outer part. The scaffolds were characterized for their mechanical properties, which resulted similar to that of the natural human coronary arteries. Moreover, the scaffolds were biocompatible, and the presence of silk fibroin increased epithelial cells viability (Yu et al., 2016).

Also in bone tissue engineering TPU has recently gained great attention, due to its excellent mechanical properties and adjustable degradation rate. Cui Z. et al. studied a 3D porous TPU scaffold enriched with HA by means of vacuum-assisted solvent casting method followed by ultrasonic technique. The HA particles on the scaffolds surface led to an increase of the systems wettability and the water adsorption capacity, both fundamental for the cell adhesion. Moreover, also the mechanical properties have been increased by the HA loading, resulting comparable to the ones of the aimed tissue. Finally, the scaffolds were characterized by great cell adhesion and proliferation (Cui et al., 2019). Similarly, hybrid electrospun fibers based on TPU and polydimethylsiloxane were enriched with nanoparticles of HA for bone reconstruction. As in the previous study, the presence of HA led to an increase in the surface wettability and hydrophilicity together with an increase in the static and dynamic mechanical properties. Moreover, the scaffolds allowed cell adhesion and proliferation and resulted highly bioactive in vitro, as they were able to promote bone-cell activity and blood compatibility (Drupitha et al., 2018). Moreover, also in the orthopaedic field TPU has showed its versatility. In fact, in a work of ours, TPU was involved for the production of electrospun aligned nanofibers enriched with chondroitin sulfate and ceria nanoparticles for tendon tissue reconstruction. The developed scaffolds were characterized by mechanical properties comparable to the ones of the native tissue. Moreover, the mechanical properties were maintained during 3 months of degradation together with the fibers' integrity, demonstrating the TPU capability to stand the stresses during the entire regeneration of the new tissue. Finally, the scaffolds allowed tenocytes adhesion and proliferation, and they resulted safe in a murine model in vivo, since they did not cause any immune response without leucocyte recruitment or foreign body response (Bianchi et al., 2023a).

In summary, TPU has emerged as a significant material in tissue engineering due to its great versatility and a wide range of favorable properties that represent great potential for its application in a wide range of fields in tissue engineering applications. However, some challenges must be also considered for its use, including degradation challenges, which could not match the rate of tissue regeneration. This aspect needs to warrant careful consideration in scaffold design and application. In fact, TPU's degradation rate must be optimized by changing the ratio between soft and hard segments, in order to avoid complications.

3.2. Polyhydroxyalkanoates

PHA are a class of natural polymers produced by different organisms. Terminologically speaking, PHA are formed by a hydroxylalkanoic acid, which can be any acid having the structure HO–R–COOH and an alkyl unit as R. According to this definition, PLA and PCL should be part of the PHA family, since they are formed by O–CH(CH₃)–COO– and O–(CH₂)₅–COO– respectively. However, in the biomaterial field PHA class is limited to the materials derived from microorganisms as lipid inclusions for energy storage in granular forms within the cellular structure, restricting this area primarily to a PHA polymer which is commercially available, the polyhydroxybutyrate (PHB), and excluding PLA and PCL (Chen et al., 2013; Drupitha et al., 2018; Steinbuchel, 2001; Lee and Choi, 1997; Williams et al., 1999).

PHB, the first and most widely studied PHA member (Fig. 8b), was discovered in 1920s in Bacillus megaterium, but its potential application in the biomedical field gained interest in the 1980s for the development of drug delivery systems (Freier, 2006). In the last decades, PHA have attracted great attention in a wide variety of medical applications, such as surgical sutures, orthopaedic uses, and cardiac substitutes, together with tissue engineering due to their mechanical properties, biocompatibility and biodegradability (Williams et al., 1999).

Fig. 8.

(a) General structure of PHA; (b) PHB structure.

As already mentioned, PHA (Fig. 8a) are produced by numerous microorganisms, including Gram–positive and Gram–negative bacteria, under conditions with limited nutrients, such as potassium, magnesium, ammonium and phosphate, and excessive availability of carbon. These unbalanced nutrients result in an excessive intracellular storage of nutrients, which are collected as PHA granules (Raza et al., 2018).

Different type of PHA can be obtained, and they are classified into three classes: short, medium or long chain length, based on the number of carbons in the side chains. The PHA with a short chain (such as PHB) are characterized by less than 5 carbon atoms, the medium chain PHA are characterized by 5 to 14 carbon atoms, while long chain PHA are formed by more than 14 carbon atoms, but they are also rare and consequently less studied (Kunasundari and Sudesh, 2011; Pulingam et al., 2022).

PHA are characterized by resistance to hydrolytic attacks, and they are insoluble in water, biocompatible and biodegradable, mostly depending on their composition. In particular, PHB shows also excellent barrier capacity, and tunable mechanical properties based on its molecular weight and level of crystallinity (Chen et al., 2013; Raza et al., 2018; Bugnicourt et al., 2014). Moreover, it also demonstrated good cell adhesion and proliferation with a wide variety of human cells, such as fibroblasts, hepatocytes, and endothelium cells. This is probably due to the fact that it has been found in cellular membranes of animals, and also in human blood (Sevastianov et al., 2003; Kaufman et al., 1983). Interestingly, PHB degradation product (3 hydrobutyric acid) has been demonstrated to be a natural human metabolite found in brain, lungs, liver, heart, kidneys, and muscular tissue (Kaufman et al., 1983). As a matter of fact, US FDA approved the use of PHA for several medical applications, for example TephaFLEX® (Carlson, 2007) and Monomax® (Odermatt et al., 2012), which are sutures made of poly(4-hydroxybutyrate) (Pulingam et al., 2022).

Similarly to TPU, PHA, and especially PHB, have been principally used in tissue engineering for the development of long-term stable implants, but that are also able to absorb. They have been studied to be applied in bone, nerve, the cardiac, urinary and gastrointestinal tract regeneration, as described in the following section.

3.2.1. Tissue engineering applications

In recent years various manufacturing procedures of PHA have been developed in order to obtain novel composite materials and overcome the disadvantages, such as the slow degradation rate and the lack of mechanical stability. Therefore, PHA have gained a great interest to be used in numerous tissue engineering fields (Pulingam et al., 2022).

Although its rigidity, scaffolds with adequate flexibility can be fabricated from PHA. In fact, these thermoplastic materials have been widely studied especially for the application in soft tissue engineering. For example, Vigneswari et al. developed a nanofibrous scaffold based on PHB combine with collagen peptides by means of electrospinning equipped with a dual syringe system. The presence of collagen led to an increase in the surface wettability. Moreover, in vitro evaluations demonstrated that the scaffolds promoted fibroblasts adhesion and proliferation, and in vivo evaluation showed that the systems of both collagen and PHB induced the healing process more than the therapy with a traditional gauze (98 % and 63 % respectively) (Vigneswari et al., 2016). In a similar work, collagen, gelatin and keratin were mixed with PHB to develop nanofibrous mats and evaluate their cytocompatibility. Fibroblasts were able to proliferate on all the matrices, and in particular the scaffolds of PHB combined with collagen showed an enhanced proliferation respect the other proteins, indicating the great potential of combining PHB with collagen for wound healing (Wang et al., 2016).

As already mentioned, PHA have also been widely studied for cardiac tissue engineering, thanks to their biocompatibility and mechanical properties. In a large animal model, Opitz et al. developed a PHB scaffold in order to regenerate a section of the descending aorta. The scaffolds mechanical properties were tested in a bioreactor, and they were shown to be similar to those of the native aorta. Moreover, the scaffolds were able to tolerate systemic blood pressure for up to 3 months, probably due to the PHB high flexibility (Opiz et al., 2004).

Due to their mechanical properties and slow degradation rate, PHA have been also investigated in hard tissue engineering, especially for bone and cartilage regeneration (Lim et al., 2017). Recently, Parvizifard et al. studied the effect of a scaffold fabricated by foam replication method and based on PHB combined with chitosan and multi-walled carbon nanotubes. The systems were characterized by adequate compressive strength, and the chitosan coating improved the systems wettability. Moreover, the results showed that the scaffolds improved cell proliferation and increased the alkaline phosphatase secretion, also due to the surface roughness which improved protein binding (Parvizifard and Karbasi, 2020). Also in another study PHB was combined with chitosan and multi-walled carbon nanotubes. Carbon nanotubes improved the scaffolds tensile strength and wettability, together with a reduction of the degradation rate. The systems also showed enhanced chondrocytes adhesion and proliferation, especially where the mechanical properties were similar to those of human articular cartilage (Mohammadalizadeh and Karbasi, 2020).

In short, PHA, and PHB in particular, are characterized by numerous properties that result favorable for the regeneration of tissues, such as mechanical properties and degradation rate. However, their production process from microorganisms is expensive, thus nowadays they are mainly used for research and development purposes.

3.3. Poly(butyl cyanoacrylate)

PBCA is a biodegradable and biocompatible homopolymer extensively studied and used nowadays in the medical field as drug delivery system to target cancer or pass the blood-brain barrier (Vauthier et al., 2003). It is a member of the poly(alkyl cyanoacrylates) family, firstly synthesized and characterized in 1979 by Couvreur et al. (1979). PBCA (Fig. 9a) is composed of n-butyl-2-cyanoacrylate (BCA) monomer units, which is also used as biomedical adhesive tissue thanks to its fast polymerization mechanism (Keller et al., 2022). Since BCA is reported as one of the most reactive monomers, the predominant polymerization method used is the anionic polymerization, where traces of weak bases (including water) initiate the process, while strong acids or superacids allow chain termination (Limouzin et al., 2003). Typically, the polymerization results in PBCA with a molecular weight lower than 3000 g/mol due to the instability of PBCA with high molecular weight. This is mainly due to the high reactivity of the monomer's chemical structure (Keller et al., 2022; Limouzin et al., 2003). The instability of the polymerization can be attributed to the β‑carbonyl-like structure of alkyl cyanoacrylates. In fact, this tends to form carbanions which are stabilized by the strong electronegativity of the nitrile and carbonyl group. Their stabilization also confers a high reactivity to the monomer enabling the polymer chain to reorganize depending on the nearby environment (Limouzin et al., 2003).

Fig. 9.

(a) General structure of PBCA; (b) BCA polymerization.

To overcome this problem, a complex pathway covering initiation, reversible propagation and reversible termination was proposed to allow the control of polymerization in an acidic medium (Fig. 9b), also confirming that the pH can be used to control the molar masses and the particle stability (Behan, 2000; Behan et al., 2001). This process has been extensively used for the preparation of PBCA nanoparticles in the medical field.

PBCA is widely studied in the medical field due to its low cell toxicity and biodegradable properties, also showing rapid body elimination. In particular, it is hydrolytically degraded in biological media yielding n-butanol and poly (cyanoacrylic acid), both soluble in water and excreted by kidney filtration (Semyari et al., 2021; Koohi Moftakhari Esfahani et al., 2016).

3.3.1. Tissue engineering applications

As already mentioned, alkyl cyanoacrylates were firstly introduced in the medical field as tissue glues for skin regeneration due to their ability of creating in situ PBCA films (Keller et al., 2022; Vauthier, 2019). In a recent work, a biocompatible and biodegradable PBCA was synthesized and used as backbone for chitosan surface modification in order to avoid the problems related to the use of chitosan alone as a carrier. The results showed that the combination of chitosan and PBCA maintained the positive characteristics of chitosan, removing issues associated with poor solubility and cytotoxicity. Moreover, the system stability against DNA digestion, endo-lysosomal escape and enhanced gene expression, shows a great versatility for the transfection-guided differentiation of induced pluripotent stem cells in a murine model (Lin et al., 2021). Another study developed PBCA nanoparticles loaded with carboplatin for the treatment of ovarian cancer. The nanoparticles were also coated with hydrophilic polyethylene glycol to improve the treatment efficacy, enhancing the system properties. The nanoparticles resulted stable over time and the efficiency towards A2780CIS and A2780CP ovarian cancerous cell lines was enhanced in respect to the free carboplatin (Hassanzadeganroudsari et al., 2019).

However, PBCA also shows bacteriostatic properties, and it can be easily handled with numerous techniques, such as vapor phase polymerization (Mankidy et al., 2009) and electrospinning, allowing also the development of nanofibrous scaffolds for tissue engineering purpose. At this purpose, Carilles et al. (2021) studied the electrosinning process of PBCA in a wide range of molecular weights in order to produce novel fibrous scaffolds with potential applications in drug delivery and tissue engineering. 5-fluorouracil was also loaded into the meshes to evaluate the release behavior of the drug in correlation with the fibers degradation rate. Micro- and nano-fibrous PBCA scaffolds were successfully developed, showing adequate tensile propertied and excellent cell adhesion and proliferation despite the PBCA hydrophobic nature. Moreover, the systems were also characterized by a slow and constant drug release, showing potential as delivery systems required for long drug release (Carilles et al., 2021). Similarly, in a work of our research group, a nanofibrous scaffold based on PBCA was developed and doped with copper oxide nanoparticles and caseinophosphopeptides, in order to improve the tendon tissue healing potential. The scaffold was developed by means of electrospinning and it was characterized by aligned nanofibers, which increased the system mechanical properties. Moreover, the scaffold resulted biocompatible towards human tenocytes, promoting cell adhesion and proliferation in vitro. More importantly, the system antibacterial activity was evaluated demonstrating its significant antimicrobial effect against E. coli. (Bianchi et al., 2023b).

In summary, PBCA is recognized for its biocompatibility and biodegradability, making it suitable for various applications, including scaffolds for tendon tissue engineering and drug delivery systems. However, there are also limitations that must be considered. For example, the mechanical properties of PBCA could not sustain long-term load-bearing applications, potentially leading to structural failure. This suggests the need for ongoing research to optimize its applications.

4. Thermoplastic polymers and mechanotransduction

It is known that tissue-specific progenitors can mature when cultured on substrates mimicking the physiological stiffness of their native tissues. For instance, neural stem cells, pre-osteoblasts, myoblasts, and adult cardiac progenitors, gain their respective phenotypes when in contact with matrices able to mimic their in vivo environments. Moreover, mesenchymal stem cells (MSCs) have been shown to be sensitive to substrate mechanics when shifting between osteogenic and adipogenic lineages (Martino et al., 2018).

In living organisms, cells are found in niches surrounded by the scaffolding ECM, which provides them with heterogeneous and dynamic mechanical cues, together with tissue-specific topography (Petzold and Gentleman, 2021). For this reason, engineered materials that mimic the physiological environment represent powerful tools for controlling cell behavior (Martino et al., 2018; Chen et al., 2018). In this context, mechanobiology is considered extremely important. This is a multidisciplinary field that combines materials science and engineering mechanics with cell and molecular biology to explore how stem cells could detect (mechanosensation) and react (mechanotransduction) to the alterations in their mechanical environment. In particular, stem cells are able to monitor their physical surroundings through macromolecular complexes known as mechanosensors. Moreover, they are able to initiate an adaptive response if the mechanical environment is unfavorable (Naqvi and McNamara, 2020). In fact, mechanotransduction is generally derived from the bonds between the cells and the ECM, applying forces through the membrane protein integrin and initiating a cascade of different pathways that alter the cells and their fate. Furthermore, the response is cell-type specific, meaning that different types of cells react in different ways to the tension applied from the surrounding ECM (Huang et al., 2019).

The principal way for the mechanical forces transmittance to the cells is filamentous actin (F-actin), which affects various processes, such as adhesion, multiplication, and morphogenesis changes. In fact, when an external force is applied, the extracellular α-chain of integrin is activated and transmits the signal to the corresponding F-actin cytoskeleton, causing an internal contractility and forming integrin-based focal adhesion complexes which bond the ECM (Harasimov and Schuh, 2018; Shou et al., 2023). Also membrane ion channels result as mechanosensitive detectors. In fact, their molecular conformation can be changed when they sense mechanical stimuli, consequently changing the membrane permeability for specific ions and altering cell fate. Specifically, G-protein-coupled receptors (GPCR) are the principal ion channels for the detection of external forces as they release signaling molecules that remodels the surrounding matrix and impact cells behavior. After they receive the mechanical signals, the gene expression is regulated through different pathways, which involve biochemical signals in the cytoplasm that are transmitted to the nucleus (Ahearne, 2014; Delmas and Coste, 2013).

An important pathway for gene-regulated mechanotransduction is the Hippo pathway, which modulates cell proliferation, differentiation, and migration (Fig. 10a). Upstream effectors, such as cell-cell interactions, lead to the phosphorylation of mammalian sterile 20-like kinase 1/2 (MST1/2), which is a serine–threonine kinase able to regulate apoptosis and proliferation, consequently maintaining tissue homeostasis (Yin et al., 2023). This switches on the Hippo, activating the core kinases of the pathway, Large tumor suppressor 1 and 2 (LATS1/2), which are heterodimeric large amino acid transporters (Nozaki et al., 2019). LATS1/2 induce the phosphorylation of the yes-associated protein (YAP) and the transcriptional coactivator with PDZ-binding motif (TAZ), transcriptional coactivators which form a complex that is phosphorylated in the cytoplasm and unphosphorylated in the nucleus. This results as the critical step of the pathway. In fact, mechanical stimulation can influence the location of YAP/TAZ, which is most likely to be found in the nucleus with high stimulation levels and in the cytoplasm with low stimulation. The phosphorylated YAP/TAZ interacts with 14–3-3 protein causing cytoplasmatic retention and ubiquitination processes mediated by proteasome degradation (Ortega et al., 2022). However, when subjected to mechanical stimuli, specific signals to membrane proteins, such as integrins and GPCR, activate RhoA transforming protein, that inhibits LATS1/2, inactivating the Hippo pathway. In this case, the unphosphorylated YAP/TAZ complex translocates into the nucleus and binds the transcriptional enhanced associate domain (TEAD), leading to gene expression mediation and increase in multiplication (Martino et al., 2018; Y.-C. Chang et al., 2019).

Fig. 10.

Schematic illustration of (a) Hippo pathway both in ON and OFF conformations, and (b) RhoA/ROCK pathway. This illustration was made using Biorender.

Ras homolog gene family, member A (RhoA) and its downstream effector Rho-associated protein kinase (ROCK) play important roles in multiple cellular processes, and they have the potential to work synergistically with the Hippo pathway (Deng et al., 2019). In fact, it is known that the RhoA/ROCK pathway is also involved in the mechanotransduction process (Fig. 10b). In this case, mechanical stimulation on the membrane proteins, such as GPCR and Integrins, activates the guanine nucleotide-exchange factor (GEF) as downstream response to the activation of Src kinases. The activated GEF stimulates RhoA by converting guanosine diphosphate (GDP) to guanosine triphosphate (GTP) (Burridge et al., 2019). At this point, Rhoa-GTP activates the effectors ROCK1/2, which lead to the phosphorylation of myosin light chain (MLC), promoting actomyosin contractility and the formation of actin stress fibers. This produces forces from the cytoskeleton, which can activate and translocate to the nucleus transcription regulators, such as megakaryocytic acute leukaemia (MAL). In the nucleus, MAL binds serum response factor (SRF), forming SRF-MAL complex, which influences gene expression and cell behavior. Moreover, RhoA/ROCK pathway leads to cytoskeleton remodeling, changing cell morphology (such as shape and size) and motility, as a mechanosensitive response to ECM stiffness (Shou et al., 2023).

Numerous studies on various cell types, such as endothelial cells (Yeh et al., 2012), airway smooth muscle cells (Shkumatov et al., 2015), and dermal fibroblasts (Razinia et al., 2017), reported the effects of the substrate stiffness and mechanical properties on cell proliferation. In particular, they reported that stiffer substrates seem to favor the cell cycle. However, contrasting results can be found in literature, due to the variability of both conditions and models in vitro. This highlights the necessity to obtain a specific stiffness range for each organ and/or tissue.

The most widely studied synthetic polymers, PLA, PCL, PGA, and TPU, offer greater processing flexibility and controllable physical and mechanical properties in respect to natural polymers. This makes them predominant scaffolding materials suitable for both soft and hard tissues providing the cells with mechanic stimuli (Carotenuto et al., 2022). The comparison between the mechanical properties of the thermoplastic polymers under study are reported in Table 2.

Table 2.

Comparison of the mechanical properties of the thermoplastic polymers under study.

Polymer	σ (MPa)	E (GPa)	ε (%)	Reference
PLA	21–60	0.35–3.5	2.5–6	Farah et al. (2016)
PGA	60–99.7	6.0–7.0	1.5–20	Farah et al. (2016)
PCL	20.7–42	0.21–0.44	300–1000	Farah et al. (2016); Dwivedi et al. (2020)
TPU	3.69–45		58.43–1520	Xu et al. (2020); Rohm and Manas-Zloczower (2023)
PHA/PHB	40	3.5-4.0	5.0–8.0	Farah et al. (2016)

σ: tensile strength; E: tensile modulus; ε: ultimate strain.

These polymers rigidity can significantly influence cell function, especially in terms of cell morphology, adhesion, and differentiation. In particular, stiffer substrates promote larger cell areas with well-defined stress fibers, enhancing cell adhesion and spreading. Conversely, softer substrates lead to smaller cell areas and less pronounced stress fibers, indicating reduced adhesion. The rigidity of the substrate affects stem cell differentiation; for instance, stem cells on stiffer matrices exhibit increased nuclear localization of YAP, a key regulator of cell fate (Ribeiro et al., 2021).

Moreover, mechanotransduction pathways are activated differently based on substrate stiffness, influencing cellular responses such as polarization and migration (Doss et al., 2020). The capability of tuning scaffolds' stiffness by tuning the polymers properties also allows for optimized control of cell behavior, facilitating specific tissue outcomes (Lu et al., 2019).

Moreover, these polymers can be modified to enhance cell-substrate interactions through various surface treatments and chemical modifications. These approaches aim to improve biocompatibility, promote cell adhesion, and facilitate cellular functions, ultimately optimizing the performance of biomaterials in tissue engineering and regenerative medicine. Moreover, the modifications aim to create biomimetic environments that promote cell adhesion, proliferation, and differentiation.

As already mentioned, surface modifications such as plasma treatment can be useful for the introduction of surface free radicals to enhance hydrophilicity and allow for subsequent graft polymerization. This could add ulterior specific functional groups that promote cell adhesion, enhance biocompatibility, and direct cell fate (Krutty et al., 2016). Also the creation of nanostructured surfaces can improve spatial arrangement and cell behavior, influencing proliferation and differentiation. In fact, topography controls cellular processes such as adhesion, morphogenesis, migration, differentiation, and apoptosis in conjunction with biochemical and physical stimuli. Well-defined topographical cues have the ability to alter cellular orientation and morphology, which can be used to alter other cellular responses (Rizwan et al., 2019).

While these modifications can significantly enhance cell-substrate interactions, it is essential to consider that excessive modification may lead to unintended consequences, such as altered cell behavior or toxicity. Balancing these factors is crucial for successful tissue engineering applications.

Martìnez-Moreno and coworkers studied and compared the development of porous systems based on PCL and TPU. They showed that the scaffold architecture combined with the characteristics of the thermoplastic polymers is fundamental to provide good cell integration and biomechanical properties, improving the biointegration of 3D constructs for the treatment of cartilage injures (Martínez-Moreno et al., 2021). TPU was also used for the production of electrospun nanofibrous scaffolds intended for the neural differentiation of rat MSCs. Results demonstrated an enhancement in cell attachment and growth. Moreover, cellular functions were ulteriorly enhanced by the external electromagnetic stimulation of the system, resulting in a higher differentiation and expression of neural markers (Pouladzadeh et al., 2018).

Rashad et al. developed a 3D printed PCL scaffold for bone tissue engineering. The scaffold supported an elongated cell morphology and enhanced the alkaline phosphatase activity, collagen I formation, and mineralization. Moreover, it was able to improve cells attachment, proliferation, and osteogenic differentiation (Rashad et al., 2018). In particular, it is widely known that stiffer substrates are able to upregulate osteogenic differentiation and mineralization compared to softer substrates. In a study on the effect of substrate stiffness on MSCs differentiation, varying stiffness (0.51–22 kPa) was achieved in the systems. Results showed that substrate modulus could effectively regulate osteogenic differentiation and alkaline phosphatase production (Mao et al., 2016).

Similarly, another study showed the possibility of refining the phenotype of cartilage generated from MSCs by manipulating material stiffness. In particular, a substrate was developed using different polyesters, PCL, PLA, and PGA having a wide range of mechanical stiffness. Results showed that substrate stiffness could affect both MSC morphology and the phenotypic development at the earlier stage of chondrogenic differentiation. Softer PCL and PLA allowed the cells to adopt a round morphology and produce hyaline-like cartilage with middle/deep zone cartilage characteristics. On the other hand, stiffer PGA system stimulated the cells to adopt a spread polygonal shape showing the potential to generate constituents of hyaline/fibro/hypertrophic cartilage (Wu et al., 2016).

5. Conclusions

The development of functional tissue replacements is an important challenge for human healthcare. At this purpose, numerous studies are currently being devoted to the development of scaffolds based on thermoplastic polymers as medical devices in the tissue engineering field of soft and hard tissues. These materials are characterized by remarkable mechanical properties, which could mimic the properties of the biological tissues, such as the tensile strength and the stretchability. Moreover, they also show good tissue compatibility and a slow degradation rate, which could result an important tool for the reconstruction of orthopaedic tissues. In particular, thermoplastic polymers could be effective as implants that require long retention time and also a great stability towards the surrounding tissues until they are adsorbed. Moreover, they have the benefit of being easily processable and they can be manufactured precisely with the required mechanical and physico-chemical properties.

Their disadvantages, such as the hydrophobicity, can also be easily avoided with the combination of thermoplastic polymers and natural polymers, in order to obtain biocompatible hydrophilic surfaces with mechanical properties that match those of the native tissues.

Interestingly, thermoplastic polymers result crucial tools to effectively stimulate MSCs differentiation, due to the regulation of the substrate stiffness that could create an adequate mechanical environment.

In conclusion, big steps have been made for the use of different thermoplastic polymers for the production of scaffolds in the field of tissue engineering. As described in this review, many studies highlighted the potentiality of these systems in improving the tissue healing potential while providing mechanical support, opening an opportunity for further clinical trials.

Funding

This research received no external funding.

CRediT authorship contribution statement

Eleonora Bianchi: Writing – review & editing, Writing – original draft, Investigation, Data curation, Conceptualization. Marco Ruggeri: Visualization. Barbara Vigani: Visualization. Carola Aguzzi: Visualization. Silvia Rossi: Resources, Funding acquisition. Giuseppina Sandri: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare no conflict of interest.

Data availability

Data will be made available on request.