Bone defects cause immense economic and healthcare concerns throughout the world. Reparation of large bone defects, as well as the restoration of the anatomical structure, appearance, and function of the injured area, has presented major difficulties [1]. At present, autologous and allogeneic bone transplantation remain the “gold standard” of bone substitution therapy in clinics. However, the drawbacks are numerous and well-known. Patients of autologous bone transplantation must undergo additional surgery, and there is a limited amount of autologous material, which in some cases may be insufficient for large-area bone defects. Additionally, there is a high incidence rate of complications such as fractures, nonunion, and infection after allogeneic bone transplantation. Therefore, it is necessary to devise new methods for engineering tissues that mimic native bone [2].

Bone tissue engineering is an interdisciplinary biomedical engineering method that can provide solutions to these complications, as it can act as an alternative to tissue transplantation [3]. This method involves the separation, cultivation, and expansion of autologous high-concentration cells in vitro, then their implantation on natural or synthetic cell scaffolds, or into the bone defect site [4]. With the gradual degradation of biomaterials, the implanted cells proliferate and differentiate, forming new bone tissue. This, in turn, helps to retain, maintain, and improve the functionality of damaged sites. Engineered bone tissues have the possibility to not only exert these functions at the time of treatment but also to continue the formation of functional tissue as needed after implantation and to further mature in situ [5]. The three elements of bone tissue engineering are “scaffold materials”, “seed cells”, and “growth factors”, the last of which can promote the growth and differentiation of seed cells ^[6][7][6,7].

Cells applied in the reconstruction of tissues and organs are collectively referred to as seed cells. In recent years, MSCs have been widely studied for use as seed cells in bone tissue engineering. MSCs, which were discovered by Friendenstein et al. in 1968, are widely distributed in almost all tissues of both fetuses and adults, including bone marrow, blood, the umbilical cord, umbilical cord blood, placenta, fat, the amniotic membrane, amniotic fluid, dental pulp, skin, and menstrual blood ^[8][9][8,9]. Bone marrow and adipose tissue are the most common cell sources of MSCs. MSCs not only have the potential of multi-directional differentiation, but in recent years, studies have shown that MSCs can promote vascular regeneration [10], reduce apoptosis [11], and inhibit the occurrence of inflammatory response [12].

The foundation for successful tissue construction is the interaction between the microenvironment and the seed cells [13]. However, the monolithic structure of the traditional scaffold limits mass absorption of nutrients and gas molecules such as ions, growth factors, and oxygen. Furthermore, the removal of cell metabolite by-products is a problematic issue, as well as the uneven distribution of cells and the extracellular matrix and necrosis within the structure [14]. Moreover, this “top-to-bottom” strategy is not adequate to flexibly adapt to variations in the shapes of some defects ^[15][16][15,16]. With the development of micro/nano biological manufacturing technology and biomaterials, a new kind of “bottom-up” modular tissue engineering technology was proposed as a way to construct three-dimensional functional tissues by combining micro repetitive functional units to curb those issues ^[17][18][19][17,18,19]. A simple but effective solution using this “bottom-up” method was put forward in the 1990s that involves introducing microspheres into a continuous matrix instead of compromising the properties of the bulk scaffold [20]. Microspheres are structures with diameters ranging from 1 to 1000 µm, and they can be categorized according to structural differences as solid microspheres, porous microspheres, or hollow microspheres. The solid and the porous types are most commonly used in seed cell culture processes, where the particulates act as bioreactors and exert therapeutic effects by modulating cell behavior [21]. Furthermore, their inherently small size, high drug loading efficiency, infusibility, and high reactivity to the microenvironment enable the hollow microspheres to be effective as a controlled drug delivery tool ^[22][23][22,23].

Microspheres have a number of unique properties compared to large pieces of material materials that make them attractive for biomedical applications. Microspheres can be fabricated from both natural and synthetic materials, and can be made to vary in shape, density, porosity, and size by applying techniques that are often compatible with the encapsulation of biologics. Microspheres for different application scenarios require different properties [24]. Microspheres can be divided into three categories: suspensions, granular and composites. In suspensions, the microspheres reside in a fluid (liquid or air), with minimal interactions between particles. When the particle-packing density increases, granular microspheres form. If microspheres are embedded within a bulky material, a composite is obtained [25]. Physical interactions between microspheres typically result in shear thinning behavior and solid consistency without chemical modification after injection. In addition, microspheres are inherently modular, because multiple microspheres populations can be mixed together to create different materials with different properties [26].

2. Fabrication of Microspheres

2.1. Emulsion Polymerization

Emulsion polymerization is a classic method for preparing microspheres. By utilizing an emulsifier and mechanical agitation, the water-soluble monomers are dispersed in water, thus forming an emulsion. Initiate polymerization is a special method of free radical polymerization [27]. The reaction system consists of a hydrophobic monomer, a water-soluble initiator, an emulsifier, and water. With water as the dispersion medium, the free radical generated by the initiator enters the monomer swelling micelle from the aqueous phase and reacts with the monomer to form a nucleus. The hydrophobic monomer then diffuses from the droplet through the aqueous phase. In the process, the free radical polymerizes with the nucleus generated by the monomer. With the steady growth of the nucleus, it eventually becomes a microsphere and precipitates until the monomer diffusion ends and the droplet disappears. However, inside the microsphere, the monomer continues to polymerize until the reaction ends. Microspheres prepared using this method have high homogeneity and monodispersity, alongside high polymerization speed and molecular weight [28]. The spherical particles are formed mainly because of the interfacial tension; however, in one study, Fab et al. used negatively charged hydrophilic acrylic acid as a comonomer and attempted to introduce the hydrophilic and hydrophobic vinyl monomer into the oil-water interface system. The emulsion interface of the water (hydrophilic monomer aqueous solution) and oil (containing the hydrophobic monomer) was subsequently constructed, and the anisotropic Janus microspheres with hydrophile lipophilic properties were successfully formed by emulsion polymerization. This method can not only synthesize anisotropic Janus microspheres, but also synthesize spherical microspheres with porous structure, offering an entirely new direction for emulsion polymerization [29]. Other types of emulsion polymerization include core shell emulsion polymerization, soap-free emulsion copolymerization, and microemulsion polymerization. Physical methods such as an ultrasonic magnetic field have been proposed, but they are still in the theoretical stage [30].

2.2. Suspension Polymerization

Suspension polymerization refers to the method of free radical polymerization in which the monomer is dissolved with the initiator and suspended in water in the form of droplets, with the water as the continuous phase and monomer as the dispersed phase. Suspension polymerization has a high polymerization rate and requires a high relative molecular mass of the microspheres, whereas the impurity content is far lower than that of emulsion polymerization [31]. Using this method, researchers have successfully synthesized nano monodisperse crosslinked polystyrene microspheres without any chemical derivation or grafting steps. The nano microspheres synthesized using this method have a good shape and no impurities on the surface [32]. In addition to smooth microspheres, porous polymer microspheres have been widely used in the immobilization of enzymes and catalysts, adsorption and separation, detection, and sensing due to their high specific surface area, abundant pores, and good stability. In one case, Zhang et al. successfully prepared surface folded porous polymer microspheres in the traditional suspension polymerization system with the help of phase separation and volume shrinkage [33].

2.3. Precipitation Polymerization

The microspheres obtained via precipitation polymerization not only have narrow particle size distribution and a simple operation process, but they are also easily obtained for follow-up treatments. Unlike other preparation methods, the precipitation polymerization process does not use an additional dispersion stabilizer, so the reaction conditions must be accurately controlled. Additionally, because of the absence of the dispersion stabilizer, and thus the absence of the need to separate it, it is safer for application in the biomedical system [34]. In their study, Yin et al. prepared functional gel microspheres with high adsorption capacity for cationic toxins through the in situ crosslinking polymerization of precipitate droplets. This preparation method is quite simple to achieve. First, a homogeneous aqueous phase reaction solution (monomer, initiator, crosslinking agent, and water) is prepared and then dropped into hot corn oil that has been heated by externally circulating water at 90 °C. Using this method, polymerization is completed in a very short time [35]. One noteworthy type of precipitation polymerization is called dispersion polymerization. Because of its unique polymerization mechanics, dispersion polymerization is widely used in the preparation of various types of polymer microspheres with a size of 0.1–15 μm in diameter [36]. Unlike other methods of precipitation polymerization, stabilizer is needed in the dispersion polymerization system. Precipitation polymerization is the formation of polymers that are insoluble in the mutual poly-sink system [37]. When the length of the oligomer chain generated by the reaction reaches a critical value, the polymer cannot remain dissolved in the reaction medium, allowing polymer microspheres to be obtained. The microspheres are characterized by having a good spherical shape, large particle size (compared with those of emulsion polymerization), narrow particle size distribution, and low viscosity. Precipitation polymerization is commonly used in the preparation of functional microspheres. The dispersion copolymerization reaction system, on the other hand, is very complex, and different comonomers form different polymerization systems, resulting in different reaction behaviors [34].

2.4. Spray Drying Method

Spray drying methods can be categorized as the pressure spray drying method, centrifugal spray drying method, or airflow spray drying method. Sprinklers, desiccators, preheaters, air separation chambers, air filters, collecting bins, and blowers are common components of spray drying. The goal of this method is to disperse the given materials as a mist of tiny droplets, expose them to a hot air flow, and evaporate most of their water, thus obtaining a powder, fine-granular finished product, or semi-finished product [38]. Quinlan et al. encapsulated vascular endothelial growth factor (VEGF) in alginate microparticles by spray drying, producing particles less than 10 µm in diameter. Using this process, they achieved effectively encapsulated and controlled VEGF release for 35 days, which was sufficient to increase tubule formation by endothelial cells in vitro [39].

2.5. Microfluidics

Microfluidics is a technology that uses microchannels to manipulate micro fluids. This method integrates one or more functions of a large laboratory on a micro or even nanoscale chip. By effectively and intelligently encapsulating cells or drugs in microspheres, the successful management of their functions and characteristics can be achieved. This process can also result in the development of some unique characteristics, which promote multi-disciplinary exchanges and cooperation, as well as the development of precision medicine, new manufacturing technology, and applied materials. In recent years, the use of microfluidic technology has shown many advantages in the preparation of microspheres. Compared with conventional preparation methods, the microfluidic preparation platform can more accurately control reaction conditions such as temperature and pressure with fast preparation speed and good reproducibility, while also using less raw materials and reagents [40]. More importantly, the size, morphology, and dispersion of particles can be accurately regulated using the microfluidics management preparation platform [41]. The main preparation methods of microspheres by microfluidics at present are the droplet template method and the flow lithography method. The droplet template method refers to the preparation of microdroplets through the introduction of fluid shear, electric field induction, centrifugal throwing, formation of microspheres in the system as a template, and proper curing. Flow lithography is a micro projection technology based on photopolymerization in multiphase laminar flow. It involves irradiating the fluid in the microfluidic chip with an ultraviolet beam in a preset shape, causing the irradiated fluid to partially polymerize according to the beam shape, thus obtaining homogeneous microspheres. The whole preparation process can be completed with the help of a commercial inverted fluorescence microscope [42]. Other studies have used sodium alginate to encapsulate single mesenchymal stem cells (MSCs) in a microfluidic platform, thus simulating a three-dimensional microenvironment to support cell viability and function, while at the same time protecting cells from environmental stresses. Compared with acellular microgels, the MSCs’ loaded alginate gel microspheres showed significant enhancement in bone formation in a rat tibia ablation model, which lays the foundation for modular bone tissue engineering [43]. In addition to the encapsulation of cells, microfluidics-controlled hydrogel microspheres provide a good supporting environment for cell adhesion and growth. In their study, Cui et al. constructed a bisphosphonate functionalized injectable hydrogel microsphere (GelMA-BP-Mg) using a metal ion ligand coordination reaction to achieve Mg²⁺ loading and controlled release via magnetics, thereby promoting cancellous bone remodeling in osteoporotic bone defects. The results of the in vivo and in vitro experiments show that the composite microspheres inhibit osteoclasts by stimulating osteoblasts and endothelial cells. This is conducive to osteogenesis and angiogenesis, which effectively promote cancellous bone regeneration [44]. Meanwhile, an efficient transfer platform was designed using new one-step microfluidic technology to achieve the combination of liposomes and photo crosslinked gel matrixes, thus forming monodisperse gel/liposome mixed microgel rapidly under ultraviolet light. Microgel can release kartogenin (KGN) continuously during the degradation period, thus providing a feasible method for the treatment of osteoarthritis in the joint release of KGN [45]. RIn this researchersview, we aim to provide a discussion on the reported particle design methods that can modulate their (bio)chemical/physical and structural characteristics (Figure 1).