Fabrication techniques involved in developing the composite scaffolds PCL/HA nanoparticles for bone tissue engineering applications

A fine-tuned combination of scaffolds, biomolecules, and mesenchymal stem cells (MSCs) is used in tissue engineering to restore the function of injured bone tissue and overcome the complications associated with its regeneration. For two decades, biomaterials have attracted much interest in mimicking the native extracellular matrix of bone tissue. To this aim, several approaches based on biomaterials combined with MSCs have been amply investigated. Recently, hydroxyapatite (HA) nanoparticles have been incorporated with polycaprolactone (PCL) matrix as a suitable substitute for bone tissue engineering applications. This review article aims at providing a brief overview on PCL/HA composite scaffold fabrication techniques such as sol–gel, rapid prototyping, electro-spinning, particulate leaching, thermally induced phase separation, and freeze-drying, as suitable approaches for tailoring morphological, mechanical, and biodegradability properties of the scaffolds for bone tissues. Among these methods, the 3D plotting method shows improvements in pore architecture (pore size of ≥600 µm and porosity of 92%), mechanical properties (higher than 18.38 MPa), biodegradability, and good bioactivity in bone tissue regeneration.


Introduction
Bone is a rigid, complex, and hierarchical structure consisting of collagen and hydroxyapatite (HA), which provides hardness and toughness to the tissue. Bone tissue consists of two different structures: an outer cortical bone, with less than 10% porosity, and an inner cancellous bone, with a porosity of 50-90%. Both structures undergo dynamic remodeling, maturation, differentiation, and resorption that are controlled via interactions among osteocyte, osteoblast, and osteoclast cells [1]. In bone remodeling, osteoblasts are primarily responsible for a new bone formation, while osteoclasts are responsible for bone resorption which is a dynamic process for maintaining a healthy bone [2]. Bone-related defects can be caused by many conditions, including trauma, tumors, and bone diseases, which cannot heal by themselves. Tissue engineering emerges as a potential approach to overcome the challenges of conventional in bone graft treatments [3]. Bone tissue engineering strategies involve a combination of scaffolds, growth factors, and stem cells to restore the function of injured tissue and overcome the complications associated with bone and other tissues repairing [4][5][6][7][8]. The scaffold should meet specific characteristics, such as physicochemical and mechanical properties, to achieve cell attachment, proliferation, and maturation, thereby enabling bone tissue formation [9][10][11][12]. The interconnected porous structure and pore size distribution are important factors to be considered for 3D scaffold fabrication, which contributes to cell penetration into the scaffold, and allowing an adequate colonization of the scaffold. A pore size with the high surface to volume ratio, as well as porosities, support cell attachment, proliferation, and osteodifferentiation by mimicking the extracellular matrix (ECM) of natural bone tissue [13][14][15]. Scaffolds with pores diameter of 100-300 µm enable successful diffusion of essential nutrients and oxygen for cell survival, and efficiently regulate the differentiation of stem cells [16][17][18][19]. Mesenchymal stem cells (MSCs) can differentiate into numerous categories of cells, which include adipocytes, osteocytes, fibroblasts, and chondrocytes. MSCs have been widely studied compared to other stem cell types for the development of engineered tissue/cell-based therapies [20,21]. The adipose tissue is the richest source of MSCs in adults, easily accessible [22]. Adipose tissue-derived MSCs lack of phenotypic characterization but they are marked by a low immunogenicity [23]. These cells are designed at the molecular level with immunophenotype properties [17]. Whereas the choice of a proper biomaterial for a three-dimensional scaffold fabrication is crucial in stimulating bone regeneration [24,25]. Scaffolds are designed to avoid immunological rejection and make them biocompatible, biodegradable, and regulate cell proliferation and differentiation, by controlling their physico-chemical properties [26,27]. Natural and synthetic polymers have been widely used as biomaterials due to their unique properties such as porosity, pore size, biokinetics, physicochemical, and mechanical properties. HA is widely used as promising osteogenic biomaterial, due to its chemical and structural similarity with mineral phase of bone ECM, along with slow biosorption [28]. Among synthetic polymers, poly-caprolactone (PCL) is a semi-crystalline polyester widely used as biomaterials in medical applications. PCL has a low melting point (55°C) and suitable properties (porosity, degradation time, and bioreabsorption) for bone tissue regeneration. PCL has a poor wetting surface and establishes weak interactions with biological fluids, preventing cell adhesion and proliferation. For this reason, HA has been incorporated in PCL matrix enhancing mechanical properties and osteogenic features of final PCL/ HA scaffolds [29,30]. This review article aims at providing a brief overview on PCL/HA composite scaffold fabrication techniques such as sol-gel, rapid prototyping, electrospinning, particulate leaching, thermally induced phase separation (TIPS), and freeze-drying, as suitable approaches for tailoring morphological, mechanical, and biodegradability properties of the scaffolds for bone tissues, but cannot be completely controlled through these methods. Among these methods, the 3D plotting method shows improvements in pore architecture, mechanical properties, biodegradability, and good bioactivity in bone tissue regeneration.
2 Methods involved in the fabrication of 3D scaffolds
Microporous structure with the pore size of 300-350 μm is formed, after soaking the scaffolds in water, where the porogen (250 μm) dissolves completely leaving behind the empty spots corresponding to pores ( Fig. 1)  and improved mechanical properties [42]. Zhu et al. prepared PCL/HA scaffolds with more than 93% porosity and 500 μm pore diameters. They were able to control the porosity based on the shape and the amount of the porogens added [43]. With the availability a wide range of porogens, it is possible to generate pore sizes of 50-400 μm with a reasonable degree of control. The scaffolds have interconnected pore structures with a pore size of 600 μm [44]. The pore size of the PCL/HA scaffold was similar to the human trabecular bone (300-1000 μm). Thadavirul et al. reported that PCL/HA composites treated with NaOH, showed a well-defined interconnected pores, increased cell proliferation, high water absorption capacity, high bone matrix deposition, and improved hydrophilicity of the scaffold [45]. A systematic study has been performed, by loading three different HA concentrations (13,20, and 26 vol %) in PCL-based composites, for improving the mechanical properties [46]. The HA nanoparticles incorporation within PCL composites increases cell differentiation, but it decreases the porosity and cell proliferation in the scaffolds [44]. In 14 days of cell culture, MTT assay, Alkaline phosphatase (ALP) activity for cell differentiation and total protein content were measured [47]. Compared to HA (13, 20, and 26 vol %), PCL/HA (90/10 vol%) showed 85% porosity with the tensile modulus of up to ±28 MPa and, increased in compression modulus and stress upto ±30 and 15.6 MPa, respectively [42]. Guarino et al. described of MSCs seeded onto porous PCL/HA composites scaffolds and cultured in an osteogenic medium for 1-5 weeks. In 3-4 weeks, MSCs were able to adhere and grow on composite substrates, but the small effect of signals on the biological response was evaluated in MSC cell culture.
To overcome this problem, a pre-osteoblastic cell line (MC-3T3-E1) has been used, showing a better cell adhesion and enhanced pre-osteoblast response on PCL/HA 3D scaffolds developed by this method. Porous PCL/HA composites are potential biomaterials for bone substitution, which exercise a beneficial influence on structural characteristics [46]. Compared to the other methods, scaffolds with interconnected networks, defined pore size, and increasing porosity can produce a greater degree of control so that the mechanical strength and the biological response can be maximized [48,49]. Although the leaching method has defined shape, salt particles led to poor interconnection during the scaffold fabrication, and this may not provide optimal scaffold permeability in vitro for cell distribution.

Sol-gel method
In the sol-gel process, the particle size is directly controlled by the interaction between calcium and phosphate with nonalkoxide, calcium nitrate tetrahydrate, ammonia, and phosphoric acid as precursor materials under controlled temperature and pH conditions ( Fig. 2

Freeze drying
Freeze-drying is a widely used conventional method for the fabrication of 3D scaffolds, making the solution to freeze at low temperature (−70 to −80°C), over the primary drying process in which the pressure is lowered through a partial vacuum in the chamber, while water and solvent in the material are removed by sublimation ( Fig. 3

Thermally Induced phase separation (TIPS)
TIPS process consists of quenching the polymer solution under the solvent freezing point and enforcing liquid-liquid separation (Fig. 4) [68,69]. In this method, HA nanoparticles were incorporated in the PCL polymer solution to make composite scaffolds (PCL/HA). HA nanoparticles were uniformly distributed in the PCL polymer matrix and observed by SEM. The PCL/HA Fig. 3 Scheme representation of composite scaffold by freeze-drying method involves the preparation of an emulsion created by homogenization of a mixture of the polymer solution, and a water phase, where the continuous phase has the polymer-rich solvent and the dispersed phase is water, quickly cooling the emulsion to catch in the liquid phase construction, and eliminating the solvent and water by freeze-drying   [71]. PCL/ HA composite scaffold was kept at −18°C for improving pore size distribution, which can be controlled by solvent phase crystallization. The HA/PCL composite scaffolds showed good osteoconductive property for bone tissue engineering applications [70]. To increase the pore architecture, Salerno et al., dissolved the polymer in an ethyl lactate/water mixture, with salt as porogen, increasing the interconnectivity and achieving a 92% of porosity [72].

Electro-spinning method
The electro-spinning technique is a simple method for 3D scaffold fabrication, based on fibrous structure, able to mimic natural ECM with an interconnected pore structure. The method helps to control mechanical properties and ensures increasing porosity (Fig. 5)    (rPCL-SiHA) and well-aligned (wPCL-SiHA), to mimic ECM and characterized by synchrotron μCT. A significant increase in the MSCs proliferation and differentiation on wPCL-SiHA, rather than rPCL-SiHA, was observed after 10 days of cell culture [80].

Rapid prototyping
Rapid prototyping technique (RP) is an advanced technique for the fabrication of well-designed 3D scaffolds with the interconnected porous structure associated with biomolecules and cells [81,82]. This techniques can fabricate composite PCL/HA scaffolds for repairing damaged bone, specifically, in the analysis of the mechanical properties [83]. Powder-based 3D printing (3DP) is an alternative method to fabricate scaffolds, but the poor mechanical properties restrict their application in bone tissue engineering. Kim  MTT and ALP studies reported improved the cell attachment and proliferation on 10% w/v PCL coated scaffold [84]. To increase the porosity of the scaffolds. Park et al., designed and fabricated composite scaffolds with a shifted pattern structure (PCL/HA/SP) by 3D plotting system to improve cell adhesion. The PCL/ HA/SP scaffold shows a good interconnected network, highly regular pore size higher than 600 μm, and porosity of 92% with well-defined geometry. The scaffold with shifted pattern had denser structure than PCL/HA and PCL. MTT assay and ALP activity resulted in an increase of cell proliferation and differentiation in PCL/HA/SP compared to the PCL and PCL/HA scaffolds. The mechanical modulus of PCL/HA/ SP is not significantly higher than the PCL and PCL/HA scaffolds [83]. Shigang Wang et al., fabricated PCL/HA composite scaffold with a porous circular structure by using 3D printing technology [85]. To further improve the mechanical properties, Liao et al., fabricated the triblock polymer mPEG-PCL-mPEG (PCL) scaffold and mPEG-PCL-mPEG/HA (PCL/HA) by solid free fabrication method [86]. The HA powder with a size of 100 μm was incorporated with mPEG-PCL-mPEG (PCL) by 0.5 weight ratio. PCL/HA biocomposite scaffold showed an increase in pore size, porosity, and mechanical properties of  [88][89][90][91][92][93]. The process solidifies PCL/HA filaments to create the porous PCL/HA composite scaffold [94]. These PCL/HA filaments were constructed by 3D design in a layer-by-layer separated by highly porous material. The addition of HA particles to the PCL polymer has shown significant increase in mechanical properties, and the ability to form apatite. The MTT and ALP studies have shown an increase in cell proliferation and differentiation of PCL/HA scaffolds [95]. Table 3 shows the morphological, mechanical, and compositional features of PCL/HA-reinforced scaffolds fabricated by employing different additive manufacturing techniques: FDM, DIW, SLS, and 3D printing [96]. Advantages and disadvantages of fabrication methods applied for generation of porous composite scaffolds PCL/ HA are shown in Table 4.

Conclusions
Tissue engineering emerges as a promising strategy to overcome the challenges of conventional bone graft treatments. PCL-based composite will be a promising biomaterial for bone tissue engineering. The combination of PCL with HA nanoparticles can result in 3D scaffold as suitable bone graft substitutes, owing to their properties such as mechanical strength, pore architecture, osteoconductivity, and osteoinductivity. PCL/HA composite scaffolds are non-toxic for cells and enhance the slow degradation, which may be suitable for bone tissue engineering. The conventional fabrication methods such as sol-gel, solvent casting, and particulate leaching, electrospinning, freeze-drying, and TIPS cannot control the pore architecture, geometry, or pore distributions of the scaffolds. On the contrary, rapid prototyping methods have been introduced to overcome the problem of conventional methods, developing customized scaffolds with high porosity, pore architecture control, ideal geometrical shape, mechanical properties, internal morphology, and mass transport properties. Specifically, the rapid prototype methods are able to fabricate the scaffolds in a layer by layer, starting from a 3D computer model of the scaffold designed with specific characteristics. The 3D plotting method is useful in the fabrication of porous PCL/HA composite scaffolds with controlled micro/macroporous structure, mechanical properties, bioresorption along with inherent osteogenic features, which are crucial for bone tissue regeneration.
Acknowledgements The authors thank National Institute of Technology, Warangal for providing research facility. SM thanks Ministry of Human Resources and Development, Government of India, New Delhi for providing fellowship. The authors thank Dr. Vishwanathan M and Santanu Sasidharan for helping in proofreading the manuscript.

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