The Application of Polycaprolactone in Three-Dimensional Printing Scaffolds for Bone Tissue Engineering

1Bone tissue, as one of the most important organs, plays multiple roles in daily life. Lots of patients are suffering from bone disease resulting from tumor resections, trauma, infections, cysts, and injuries caused by accidents. It has been reported that over four million operations using bone grafts are performed each year to treat bone defects. Autogenous bone transplantation and replacement are the main traditional options for patients with bone defects. However, the potential risks of tissue grafts including complications and secondary injuries remain a major clinical challenge.

2To overcome this shortage, bone tissue engineering is one of the most proposing alternative methods. Bone tissue engineering focuses on the main processes including cell growth and the customized construction of human bone tissue. Further, 3D printing has multiple advantages, including precise deposition, cost-effectiveness, simplicity, and cell distribution controllability. The developments and applications of 3D printing have been increasing constantly over the past few years.

3In the field of bone tissue regeneration, polycaprolactone (PCL) is one of the most common materials in fabricating scaffolds. PCL is a Food and Drug Administration (FDA) approved linear polyester with good biocompatibility, slow degradation rate, less acidic breakdown products in comparison to other polyesters, and has the potential for loadbearing applications. The slow degradation of PCL allows time for bone remodeling and can also be manipulated to adjust the polymer’s biodegradation rates. Additionally, PCL is one of the most preferred polymers for extrusion-based 3D printing due to its melting temperature of 55–60 °C. It exhibits good mechanical properties with high flexibility and great elongation, conducive to the preparation of scaffolds for craniofacial bone repair. However, pure PCL has no osteogenic potential to induce bone regeneration. Thus, researchers combine PCL with various polyesters, inorganic substances, metal elements, or collagen to improve the properties of the scaffolds. This review discusses and summarizes recent advancements in PCL-based composite scaffolds, focusing on the fabrication and functionalization methods and their application to promote bone growth in vitro and in vivo. Further, the future trends and potential clinical translations will be discussed.

4The availability of desired properties for 3D printed scaffolds relies on the printing technology that is used. Generally, 3D printing is a process of layer-by-layer fabrication using powder, liquid, or solid material substrates. Starting from the bottom and building up, each newly formed layer is triggered to adhere to the previous layer, gradually increasing the size of the construct. The techniques using in 3D printing include stereolithography (SLA), selective laser sintering (SLS), and fused deposition modelling (FDM). Because of the themoplasticity of PCL, the most common technique used for 3D printing is FDM. FDM uses a temperature controlled printhead to deposit thermoplastic material onto a platform in a layer-by-layer manner to build up a 3D construct. PCL begins to melt by being driven into a heated printhead, allowing thin layers to be deposited precisely and sequentially. The molten PCL cools in the air of the printing environment, allowing it to rapidly fuse together to create a scaffold. However, the elevated temperatures limit the inclusion of biomolecules and hydrogels.

5Multiple studies have focused on cell behaviors on PCL scaffolds the scales of which usually range from hundreds of microns to millimeters fabricated by FDM. However, there is a limitation of the techniques that can be used for the fabrication of fibrous micro-environments to study cell behaviors. Electrohydrodynamic (EHD) printing, also named Melt Electrospinning Writing (MEW), is a recently developed technology to overcome the above limitations. As shown in Figure 1, the design of MEW devices combines the advantages of conventional electrospinning and 3D printing. This technology allows micron to sub-micron fiber fabrication. Kim fabricated fibrous scaffolds with the EHD technique and demonstrated significantly high metabolic activity and mineralization of the cells cultured on the micro-fiber PCL scaffolds. The comparison of PCL scaffolds fabricated by FDM and by MEW is shown in Figure 2. The micro-fiber PCL scaffolds printed by MEW directly affected the cell adhesion morphology. SLS is another technique for PCL-based scaffold fabrication. The basic design of a SLS printer is a housing that has a powder bed, a laser, a piston to move down in the vertical direction, and a roller to spread a new layer of powder. The computer-controlled laser beam sinters the powder, and the remaining powder works as a structural support for the scaffold being constructed. With laser assistance, SLS is more accurate but more expensive than FDM.

6Bioprinting is another advanced technology which has aroused wide interest in recent years. Bioprinting can be used to deposit living cells and other biomaterials to build complex tissue constructs. For the bioprinting of PCL-based composite scaffolds, researchers usually combine PCL with hydrogels that load living cells. Bioprinters have multiple print nozzles, one for PCL scaffolds printing, and the others for cell-loaded biomaterial printing simultaneously or separately. With this technology, mesenchymal stem cells (MSCs) or human umbilical vein endothelial cells (HUVECs) were usually loaded on the scaffolds to improve the vascularization of the printed structure. The comparison of the 3D printing techniques for PCL-based scaffold fabrication is shown in Table 1.

7Numerous scaffolds produced from a variety of biomaterials have been used in the field in attempts to regenerate different tissues and organs. Generally, 3D scaffolds are designed to imitate the extracellular matrix (ECM). These scaffolds are required to possess bioactive characteristics as follows and as reported: a porous structure for the transport of nutrients, waste products, and for the communication with other cells; good biocompatibility with the controlled degradation and absorption rate of cell/tissue growth in vitro and/or in vivo; suitable surface chemistry stimulating cell ingrowth, cell attachment, and cell differentiation; and properties matching the individual clinical environments of bone defects.

8Owing to its brilliant biocompatibility and easy processability, PCL has been extensively used in scaffold fabrication. However, the poor hydrophilia and low bioactivity of pure PCL systems limit their applications in the biomedical field. Combining the PCL matrix with bioactive inorganic particles as fillers provides a promising way to overcome these shortcomings. Metals, oxides, polymers, and carbon-based materials have all been applied to PCL scaffolds for property improvement. A summary of recent researches and the advances in PCL-based composite scaffolds and property improvements is shown in Table 2.

9The effects of different materials on the performance of composite scaffolds were also compared. Ethan focused on the comparison of PCL-based scaffolds combined with tricalcium phosphate (TCP), hydroxyapatite (HA), Bio-Oss (BO), or decellularized bone matrix (DCB). They concluded that PCL-BO and PCL-DCB hybrids were superior to PCL-HA or PCL-TCP blends for bone healing applications. Marco found that different diameters of hydroxyapatite blended in the printed scaffolds had distinct performance. PCL-nano-HA scaffolds showed higher levels of alkaline phosphatase activity compared to PCL-micro-HA structures. Differing from physical mixing process, Chen synthesized poly(l-lactide-co-caprolactone-co-acryloyl carbonate)(poly(LLA-CL-AC)) by ring-opening polymerization in the presence of Sn(Oct)2 as a catalyst and octanol as an initiator for the first time. They found that the stiffness of the scaffolds increased after UV irradiation cross-linking.

10The standard approach in bone tissue engineering is to seed and grow cells on scaffolds in vitro. Typical scaffolds are 3D porous structures temporarily mimicking the natural extracellular matrix of bone. Ideally, 3D scaffolds should be highly porous, have well-interconnected pore networks, and have consistent and adequate pore size for cell migration and infiltration. Scaffold architecture design can significantly influence both mechanical properties and cell behaviors. The common structures designed for printed scaffolds are shown in Table 3.

11A lot of researchers have focused on the outer morphology of printed scaffolds. Cylindrical and cube-shaped structures are common 3D printing shapes in preliminary studies. Similarly, circular, sinusoidal, and conventional orthogonal models were also fabricated and compared in previous studies, as shown in Figure 3. The results demonstrated that less orthogonal elements enhanced osteogenic performance. Further, the scaffold shapes are usually designed to match the shape of bone defect area for clinical application.

12Numbers of studies have examined the inner structure inducement on cells behavior. First, the different deposit angle, which usually has an effect on mechanical property and porosity of scaffolds, has been studied. The scaffold porosity is an important factor affecting the performance of scaffolds. Shim found that 3D printed PCL GBR membranes with 30% porosity (130 μm pore size) were excellent for calvarial regeneration. Second, the pore structure has also been studied. Yang developed compatible scaffolds which included macropores, medium-sized pores, and small pores, and these scaffolds are tailored to be similar to that of natural extracellular matrix (ECM). Adeola focused on the effects of pore geometry on modulating mechanical behavior of PCL scaffolds. Lee found that the kagome structure obviously improved the mechanical properties of PCL scaffolds compared to the grid structure. Abigail fabricated artificial models that mimic the microstructure of bone, improving the accuracy of bone grafts.

13In the field of tissue engineering, personalized medicine highlights the use of specifically designed scaffolds. The customized scaffolds can optimize the repairing process in cases of irregular-shaped wounds and tissue defects, especially for orthopedic, oral, and maxillofacial surgery. With the aid of computed tomography (CT) or magnetic resonance imaging (MRI) data, the fabrication of patient-customized scaffolds using 3D printing technology is realizable. In this way, the mechanical properties, pore size, and porosity of the scaffold can be controlled. Bae successfully conducted the implant process using PCL-based scaffolds in the beagle model (Figure 4). These scaffolds restored the original volume and shape of the alveolar ridge in the defect site and performed well as a surgical guide to place the implant at the proper location and depth.

14Naturally based hydrogels can offer excellent cellular interaction and biocompatibility, but they suffer from poor mechanical properties. On the other hand, PCL printed scaffolds possess compressive strengths within the range of cortical bone. This has aroused great interest in the 3D bioprinting of cell laden hydrogel bioinks reinforced with stiffer PCL fibers. Combining these two materials, cells can be diffused on the scaffolds accurately with the bioprinting process. Researchers demonstrated that the cells which were injected into the pores of scaffolds before clinical implantation, indicating promising osteogenesis enhancement in vivo.

15There are a number of hydrogels that can be used for cell loading medium, such as matrigel, alginate, agarose, hyaluronic acid, and GelMA. In the study of Caroline Murphy, they presented a scaffold with a mixture extrudable paste of PCL and borate glass. Human adipose stem cells suspended in matrigel were then ejected inside of the scaffold as droplets. They found a controlled release of the bioactive glass for up to 14 days with degradation of the scaffolds. The results showed a high level of angiogenesis in the interior the scaffold. Stichler prepared hyaluronic acid/poly(glycolic acid) mixed hydrogels using UV light cross-linking. After loading human and equine mesenchymal stem cells, the PCL-based composite scaffolds were prepared using double-nozzle 3D printing technology. The cells showed a good chondrogenic differentiation prospect after 21 days. The combination of PCL and hydrogel improves the mechanical properties. At the same time, the existence of hydrogel is more suitable for the growth and reproduction of cells than pure PCL. Due to the high plasticity of PCL and the advantages of 3D printing technology, construction of personalized biomimetic tissue has become practical.

16It has been well-established that lack of vascularization within the engineered bone grafts is a major barrier to bone healing. Aiming to overcome this problem, Mitchell presented a hydrogel-based prevascularization strategy to generate prevascularized bone scaffolds. They coated co-culture PCL/HA scaffolds with hydrogels, which encapsulated ADMSC and HUVEC. This co-culture system promoted vascularization in vitro and in vivo. Similarly, Wen fabricated a PCL/polydopamine-modified calcium silicate scaffold loading with ADMSC and HUVEC. Xie incorporated mesenchymal stem cell-derived microvesicles into alginate/ PCL constructs for angiogenesis promotion.

17Scaffold bioactivity can be increased by adding components that are able to interact with or bind to living tissues. For a better clinical effect, PCL-based scaffolds with therapeutic agents added during the 3D process can be used as a carrier to load drugs and other bioactive substances to realize a long-term release.

18Multiple studies have investigated the pharmacokinetic of 3D printed scaffolds. Hoang used porogen leaching and 3D printing techniques and created microporous PCL scaffolds with micropors for drug loading and releasing control. They found that microscale porosity avoided the burst release of drugs and maintained relatively long-lasting drug concentrations. A summary of drug loading in PCL-based composite scaffolds is presented in Table 4.

19Biomaterials are designed to release bioactive substances at the injury site to stimulate bone repair. The modification of bioactive components on 3D scaffolds, which enable bone cells to function in a sustainable manner, has aroused great interest worldwide. To mimic the physiological bone hierarchy, the bioactive molecules were added to PCL-based scaffolds for osseointegration (Table 5).

20Bone morphogenetic proteins (BMPs) are considered to be the most eminent in advancing bone growth by inducing osteogenic differentiation. In Jang’s study, they made alginate/BMP-2/umbilical cord serum (UCS) coated on 3D-printed PCL scaffolds and demonstrated that the simultaneous use of low-dose BMP-2 and UCS significantly increased osteogenesis based in vitro and in vivo. To promote successful bone regeneration, efficient vascularization is a pre-requisite. Therefore, the angiogenic growth factor VEGF and its controlled delivery play a vital part in bone regeneration. Additionally, Eric constructed PCL scaffolds seeded with microspheres containing VEGF and VEGF with either BMP-2 or FGF-2 and observed significantly higher vascular ingrowth and vessel penetration than the controls. Collagen type I (COLI) can also be coated to the 3D printed structure, promoting the proliferation of chondrocytes. Moreover, Won compared the ability of promoting cell activity and mineralization between rhBM-2 and platelet-rich plasma (PRP) in scaffolds and concluded that rhBMP-2 was more efficient. As for mental molecule modification, gold nanoparticles (GNPs) grown on the polydopamine (PDA) coating of scaffolds have been demonstrated to be effective for bone regeneration. Further, Michal confirmed that osteoclast activity was greatly suppressed by the lithium release of printed PCL scaffolds.

21For clinical application, three types of clinical applications for 3D-printed technologies are defined in previous studies. The first is for prosthetic rehabilitation (improve patient aesthetic), the second is for reconstruction (tissue grafting), and last is for tissue regeneration (recapitulate native tissue structure and function). PCL-based scaffolds can be used in many kinds of tissue engineering, such as skin regeneration, skeletal muscle tissue regeneration, and tendon regeneration. In the field of bone defect therapy, there are reconstruction and regeneration processes for cartilage tissue and bone tissue.

22PCL is reported as a popular material in cartilage regeneration. Combining PCL with agarose and GelMA or MECM, engineered meniscus were constructed, which had the potential for use as a substitute for total meniscus replacement. Zhang and his colleagues constructed scaffolds for total meniscal substitution in a rabbit model. With the aid of multidetector CT and computer design, 3D scaffolds for use in total ear reconstruction were successfully fabricated. Additionally, 3D printing techniques were also used for trachea engineering. For instance, Parket, Shan, and Gao have successfully fabricated trachea scaffolds. Interestingly, Parket implanted the tracheal scaffold into the omentum before tracheal scaffold implantation in rabbits and concluded that the omentum-culture of the tracheal scaffold was beneficial for rapid the re-epithelialization and revascularization of the scaffold. Further, it also prevented postoperative luminal stenosis.

23For animal surgery, Carla conducted a surgical therapy of a chronic oronasal fistula in a cat using autologous platelet-rich fibrin and bone marrow loaded printed PCL scaffold. A CT scan revealed complete healing after a six-month follow-up. In Lee’s research, a customized scaffold matched with an 8-shaped bone defect on the rabbit calvarium model was designed according to 3D computed tomography. they then implanted the scaffold in the defect area, which showed excellent mechanical robustness and enhanced osteoconductivity. Rebecca Chung proposed a patient-specific 3D printed bioresorbable graft substitute for segmental bone replacement.

24In the dental application field, it is difficult to fabricate a scaffold matching the complex shape and functions of the nature craniomaxillofacial (CMF) bones. With the CT multidetector data, we can construct patient-customized structure 3D scaffolds for bone tissue regeneration using 3D printing. Joshua printed an anatomically shaped scaffold that closely resembled the 3D models. A custom scaffold can be used simultaneously as an implant surgical guide and as a bone graft in a large bone defect site. Upon dental implant surgery, successful implant placement is reliant on adequate alveolar bone volume at the implant site, which can provide mechanical stability for dental implants. It is important to augment the alveolar ridge for enabling the placement of dental implants and thus to restore both functionality and esthetic appearance. Rider and Vaquette proposed that printed scaffolds showed potential in transferring to alveolar vertical bone augmentation (Figure 5). Due to the high compressive strength of the printed structure, these scaffolds may be applicable for procedures involving simultaneous implant placement and ridge augmentation.

25PCL is a common polymer with unique biomedical and mechanical properties that make it favorable for a wide range of bone tissue engineering applications. Its low degeneration allows the imperative periods needed for new bone regeneration. With the development of three-dimensional printing techniques, various 3D structures can be fabricated successfully. Numbers of materials were utilized in the studies on PCL-based composite scaffolds, and composite scaffolds demonstrated superior performance to pure PCL scaffolds in recent studies. Cells can be also printed into the scaffolds by blending with hydrogel, which provides a compatible medium for cell proliferation. Further, in vivo studies of PCL scaffolds used in bone or cartilage tissue engineering applications have proven their osteogenic potential. Nevertheless, these studies were mostly conducted in small animals (usually rats and rabbits), which may not sufficiently predict clinical application in humans. Almost none of the researchers have proceeded to the phase of human trials yet. Thus, the advantages of PCL-based tissue engineering remain distant for patients in hospital. However, PCL is still a promising biomaterial. Future work should focus on PCL-based scaffolds in large animal models as well as in human clinical trials. We expect to develop custom-made 3D composite scaffolds that can be grafted directly with stem cells in clinical practice.