Tissue engineering is promising to meet the increasing need for bone regeneration. Nanostructured calcium phosphate (CaP) biomaterials/scaffolds are of special interest as they share chemical/crystallographic similarities to inorganic components of bone. Three applications of nano-CaP are discussed in this review: nanostructured calcium phosphate cement (CPC); nano-CaP composites; and nano-CaP coatings. The interactions between stem cells and nano-CaP are highlighted, including cell attachment, orientation/morphology, differentiation and in vivo bone regeneration. Several trends can be seen: (i) nano-CaP biomaterials support stem cell attachment/proliferation and induce osteogenic differentiation, in some cases even without osteogenic supplements; (ii) the influence of nano-CaP surface patterns on cell alignment is not prominent due to non-uniform distribution of nano-crystals; (iii) nano-CaP can achieve better bone regeneration than conventional CaP biomaterials; (iv) combining stem cells with nano-CaP accelerates bone regeneration, the effect of which can be further enhanced by growth factors; and (v) cell microencapsulation in nano-CaP scaffolds is promising for bone tissue engineering. These understandings would help researchers to further uncover the underlying mechanisms and interactions in nano-CaP stem cell constructs in vitro and in vivo, tailor nano-CaP composite construct design and stem cell type selection to enhance cell function and bone regeneration, and translate laboratory findings to clinical treatments.
Bone fracture is a substantial public health issue, and the need for bone regeneration is increasing dramatically as the world population ages.1,
The use of nanostructured biomaterials in bone regeneration is inspired by the native bone architecture. Bone possesses a complex organic–inorganic nanocomposite structure. The organic phase is mainly composed of type I collagen, which is arranged into nanofibers ranging from 50 to 500 nm in diameter.11 The inorganic phase consists of non-stoichiometric hydroxyapatite (HA) crystals with lengths of about 100 nm, widths of 20–30 nm and thicknesses of 3–6 nm, which are embedded between the collagen fibers.12,13 In 2011, the European Commission adopted the following definition of a nanomaterial: ‘A natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range of 1 nm–100 nm’.14 Some literatures more broadly use the prefix ‘nano-’ to also include structures slightly exceeding 100 nm to a few hundred nanometers. It is of great interest to develop biomimetic nanostructured scaffolds to mimic native bone. Calcium phosphate (CaP) biomaterials are of special interest because they mimic the major inorganic component of bone, are bioactive and can form intimate and functional interfaces with neighboring bone. Various forms of CaP are widely studied for bone regeneration research. Commonly used CaPs include monocalcium phosphate monohydrate, monocalcium phosphate anhydrous, dicalcium phosphate dihydrate, dicalcium phosphate anhydrous, octacalcium phosphate, α- and β-tricalcium phosphate (TCP), amorphous CaP (ACP), calcium-deficient hydroxyapatite and HA.15 Using different synthetic methods, nano-CaP crystals with diverse structures have been fabricated including particles, spheres, rods/needles/wires/fibers/whiskers, sheets/disks/flakes/platelets/strips and various 3D architectures.16 Importantly, several reports showed that nano-CaP biomaterials exhibited physicochemical and biological characteristics better than conventional-sized CaPs, due to nano-CaPs being more similar to bone nanocrystals.17,18 Thus, nano-CaPs have great potential in bone repair and augmentation.
A number of recent reviews on nanomaterials covered topics on materials selection (metals, ceramics, polymers and composites)19 and synthesis and processing techniques (e.g., blasting, etching and anodization for metals; electrospinning, phase separation, self-assembly and precipitation for ceramics/polymers/composites; and lithography, laser ablation and nanoimprinting for nanotopography).20,21Other reviews covered nanotopography22 and its influence on cells.23 Others reviewed nanopolymers (natural and synthetic polymers)24 and nanocomposites (e.g., biopolymers, polymer/ceramic, metal/ceramic).25 Still other reviews focused on nanofibers,26 nanoparticles27 and multiple applications in regenerative medicine (e.g., tissue engineering, cell therapy, diagnosis, and drug and gene delivery).28 To avoid repetition, the present review focuses on nanostructured CaP biomaterials, highlighting their interactions with stem cells including cell attachment, orientation and morphology, osteogenic differentiation, as well as cell encapsulation and delivery for in vivo bone regeneration.
Nanostructured calcium phosphate biomaterials
Nanostructured calcium phosphate cement (CPC)
CPCs are self-setting synthetic bone graft materials.29,
Another strategy to obtain a nanostructured CPC is to reduce the starting particle size of CPC to the nanoscale level. Brunner et al. used a flame-spray synthesis method to prepare amorphous TCP nanoparticles.47 Due to the higher surface area, amorphous TCP nanoparticles significantly accelerated the setting time and the conversion to apatite during the self-hardening of CPC. The addition of nanoparticulate amorphous TCP favored the nucleation of smaller crystals and promoted the formation of nano-apatite crystals (100–200 nm) in CPC.15,47
Nanostructured CaP composites
Composite approaches can be used to improve the mechanical properties of nanostructured CaP in order to satisfy clinical needs in load-bearing areas. Combining natural or synthetic polymers with nanostructured CaP is a promising strategy, since bone tissue itself is a nanocomposite of HA and collagen. Many degradable polymers have been explored for this purpose, such as collagen fibers,48 silk fibrion,49 gelatin,50 chitosan,51 poly-L-lactide,52 poly-DL-lactide-co-glycolide (PLGA)53 and poly(vinylalcohol).54 The compositions and properties of several recently-developed nanostructured CaP composites are briefly reviewed in Table 1. Each type of polymer has its own characteristics to contribute to the property improvement of the composite. Collagen is the most abundant polymer in bone tissue. By incorporating collagen into the composite, it provides more cell recognition sites and accelerates biomaterial’s degradation rate, thus allowing fast replacement by new bone.48,55,56 However, the use of collagen is limited as it is costly, and its potential of antigenicity and pathogen transmission.48,55,56 Gelatin is a denatured form of collagen, which is free of immunogenic concerns. Gelatin contains integrin binding sites which are important for cell adhesion.50 Other natural polymers such as chitosan and silk are especially known for their excellent mechanical properties.49,51 Synthetic polymers represent another category, with the main advantage of avoiding immunogenicity and disease transmission, and possessing flexibility in property controls.52,
Nanostructured CaP coatings
Another important application of nanostructured CaP is coating on metallic or other implants to enhance the bioactivity and osteoconductivity of bioinert materials. CaP can facilitate osteointegration with natural bone through the formation of an apatite layer.21 In addition, nanoscale modification of implant surfaces has been suggested to further increase its biomimicry and bioactivity.59 Nanostructured CaP coatings can be prepared by a biomimetic coating method,60 sputter deposition,61 ectrochemical deposition62 and plasma-spraying methods.63 CaP coatings with different compositions (e.g., HA, brushite or apatite),64,65 crystallinity (amorphous or crystalline)66 and surface features of the substrate (e.g., polished or roughened)60 can all affect osseointegration in vivo. Figure 2 shows schematically the biological response of a nanostructured ACP (nACP)-coated titanium implant.66 In an aqueous medium, nACP can readily release Ca2+ and PO43− ions. They can bind with serum proteins that facilitate cell attachment and subsequent formation of apatite on the implant surfaces.66 To decrease the unfavorable effects of rapid dissolution of the nACP coating, more stable CaP phases such as poorly-crystalline nano-apatite or highly-crystalline nano-HA (nHA) can be used.66 Poorly-crystalline nano-apatite showed a favorable effect on the osteogenic differentiation of osteoblasts.66
Stem cell interactions with nano-cap biomaterials
An ideal orthopedic repair material is more than just fillers for bone defects. It also serves as a scaffold to provide chemical, mechanical and topographical cues to regulate cell behavior. Many studies revealed that nanostructured biomaterials promoted the process of bone regeneration by supporting cell adhesion, spreading, proliferation and differentiation.23,67 Understanding the interactions between stem cells and nanostructured materials is of utmost importance in designing and fabricating novel biomaterials that can guide cell behaviors in a desirable way. The present review article focuses on recent studies on the interactions between stem cells and nanostructured CaP scaffolds for bone tissue engineering both in vitro and in vivo. For biomaterial interactions with other cell types (e.g., fibroblastic cells, endothelial cells, epithelial cells and macrophage cells) and nanomaterials other than CaP-based biomaterials, several review articles already exist.23,67
Previous studies investigated the behavior of various types of stem cells attaching to nanostructured CPCs with different compositions. All the tested stem cells, including rat68 and hBMSCs,62 hUCMSCs,63,69 hESC-MSCs64 and hiPSC-MSCs,65 attached well to CPC scaffolds containing apatite nanocrystals. Scanning electron microscopy examination showed that the cells anchored to nano-apatite crystals via cytoplasmic processes and exhibited a healthy spreading polygonal morphology (Figure 1b). The cytoplasmic extensions are crucial for cell adhesion, migration and formation of cell–cell junctions. The nano-apatite surfaces favorably supported proliferation of these MSCs. In another study, a nanofiberous scaffold with gradients in amorphous calcium phosphate nanoparticles (nACP) was fabricated by a two-spinnerette electrospining.72 The adhesion and proliferation of MC3T3-E1 murine pre-osteoblasts was enhanced in the gradient regions, indicating that higher nACP content yielded a better cell response.72 Therefore, stem cells can have strong interactions with nanostructured CaP biomaterials, and nanostructures can be tailored to guide and enhance cell function.
Another key aspect is cell orientation and morphology. Nano-sized surface structures provide important cues to regulate cell orientation and morphology.23,67 Among the various two-dimensional topographical nanoscale features (e.g., grooves, pits, wells, steps, pillars, pores, ridges, etc.), nanogrooves appeared to provide the most powerful and clear cues in regulating cell orientation and morphology.23,67 Regardless of different substrates and cell types, in most cases, cells aligned their shape and elongation in the long axis of the grooves, with the organization of actin and other cytoskeletal proteins to be parallel to the grooves.23 However, since nano-CaP is less maneuverable in fabrication than polymers or metals, it is more challenging to produce highly patterned nano-CaP surface features. Irregular surface geometry due to the non-uniform distribution of nHA may not promote cell orientation in a polarized direction. Hence, there are few studies reporting the particular alignment patterns of cells on nano-CaP implants. However, the addition of fibers can help induce directionality for the cells. For example, Jose et al.73 produced an aligned nanofibrous PLGA/collagen/nHA scaffold with fiber diameters of 100–350 nm. hMSCs assumed an aligned morphology along the direction of the fiber orientation.73 Electrospinning is a versatile technique to produce aligned nanofibrous scaffolds. By controlling the spinning conditions, parameters, and compositions of the solution, oriented fibers with different compositions and diameters can be obtained. The aligned nanostructures of the biomaterial can effectively induce an aligned morphology for most types of seeded cells.74
Nanostructured materials also have significant effects on cell differentiation. There is growing evidence indicating that nanofeatures are a compelling determinant of stem cell lineage commitment.75,
Notably, hMSCs behaved differently in response to different scales of CaP crystals and topography. In one study, three types of chitosan/CaP films with different surface features were fabricated by a solvent-casting method.93 The proliferation of hMSCs cultured on the nanocrystalline CaP/chitosan film was higher than that on nano-amorphous or microparticle CaP/chitosan films. Osteogenic differentiation achieved the highest level on the scattered microparticle CaP/chitosan film.93 Similar observations were reported by Garcia-Gareta et al.,60 showing that nano-sized CaP crystals and relatively flat topographies enhanced MSCs proliferation, while rougher, microscale topographies enhanced osteogenic differentiation of MSCs. Both of these reports indicated that nano-sized CaP features enhanced cell proliferation, while micro-sized CaP composites enhanced the osteogenic differentiation of the cells.60,93
In vivo bone regeneration
Several in vivo studies have shown favorable bone regeneration via nanostructured CaP biomaterials. The effect of a composite’s particle size on alveolar bone reconstruction was studied in a rat osteoporotic alveolar bone model.53 Micro- and nanoparticulate CaP/PLGA composites were applied to restore bone defects (1.6 mm in diameter and 1.8 mm deep). The new bone amount generated by the nanoparticulate CaP/PLGA group was more pronounced than the micro-particulate CaP/PLGA group. Adding autologous plasma further promoted the bone regeneration, with the best bone regeneration via the nano-particulate CaP/PLGA/plasma composite.53 Fricain et al., compared the bone regeneration potential of scaffolds composing of natural hydrophilic polysaccharides pullulan and dextran, supplemented with or without nHA in five animal models.87 This study included two heterotopic implantations (subcutaneously in mice and intramuscularly in goat), and three orthotopic models of critical-size defects in three different bony sites (femoral condyle of rat, a transversal mandibular defect and a tibial osteotomy in goats). The results indicated that only the nHA-incorporated scaffold induced a biological apatite layer and favored the formation of a dense mineralized tissue subcutaneously in mice, as well as osteoid tissues after intramuscular implantation in goats. The addition of nHA induced early bone regeneration in all three orthotopic models regardless of the site of implantation.87 These phenomena clearly demonstrated that new bone formation via nano-sized CaP composites is superior to conventional-sized counterparts in vivo.
Beneficial in vivo effects can be further obtained by seeding stem cells to nanostructured CaP materials. When a nHA/collagen I nanocomposite was enriched with hMSCs for subcutaneous implantation, the time it took for the development of a bone matrix was shortened from 2 weeks to 1 week.79 In another study on ectopic implantation, nHA/CS/PLGA scaffolds combined with pre-osteogenic hUCMSCs achieved the most bone regeneration.96 Chai et al. seeded human periosteum-derived cells onto 3D-functionalized porous nCaP–Ti6Al4V hybrids.97 These hybrids induced ectopic bone formation, which was highly dependent on the physicochemical properties of the CaP coating in a cell density-dependent manner. Only a small amount of new bone spicules was found around the hybrids when one million cells were seeded, while substantial bone formation was observed throughout the whole hybrids when three million cells were seeded.97 Besides seeding stem cells, the co-application of growth factors in nano-CaP scaffolds can further enhance bone regeneration. For example, in a rabbit calvaria bone defect model, the triple application of MSC/nHA/platelet-rich growth factor yielded 29.45% and 44.55% of new bone area at 6 and 12 weeks, respectively.98 In contrast, bone formation in nHA, nHA/platelet-rich growth factor and nHA/MSC at 6 and 12 weeks were 11.35% and 32.53%, 29.10% and 39.74%, and 25.82% and 39.11%, respectively.98
Therefore, combining stem cells with nano-CaP scaffolds can greatly enhance bone regeneration. Regarding the types of stem cells, hBMSCs are the gold standard in stem cell-based bone regeneration and have been successfully used in clinics.99 However, the use of hBMSCs is hampered by an invasive procedure to harvest, limited availability, donor site morbidity, and loss of potency of stem cells obtained from seniors and patients with diseases and disorders.100 Therefore, other stem cell sources are critically needed for bone regeneration. Currently, there are three broad types of stem cells: adult stem cells, ESCs and iPSCs. Adult stem cells can be found in stem cell niches such as bone marrow, adipose tissue, umbilical cord, placenta, etc. They exhibit a lineage-restricted differentiation potential and exhibit multipotency.101 In contrast, ESCs and iPSCs can give rise to cell types of all three germ layers, which is known as pluripotency. This feature makes these stem cells an extremely valuable and potent cell source. ESCs are obtained from the inner cell mass of blastocysts, while iPSCs are derived from reprogramming of somatic differentiated cells.102 hESCs involve complex cell culture procedures, risks of tumorigenicity and ethical concerns. Patient-specific hiPSCs with similar proliferation and differentiation ability to hESCs has the potential to overcome some of these hurdles. Thus, iPSCs are potentially the new frontier for cell-based regenerative medicine research. Several studies focused on the comparison of various types of stem cells in combination with nano-CaP biomaterials to repair bone defects. Chen et al.103 compared the in vivo bone regeneration efficiency of hUCMSCs and hBMSCs on a nano-crystalline CPC–chitosan–RGD scaffold in a nude rat cranial bone defect model. It was found that the bone-forming ability of hUCMSC–CPC constructs matched that of hBMSC–CPC constructs. 103 Our recent study revealed that, when seeded on macroporous CPC scaffolds, hiPSC-MSCs had good osteogenic capability comparable to hUCMSCs and the gold-standard hBMSCs (Figure 3). After implanting stem cell–CPC constructs in critical-sized cranial defects in rats, the new bone area fraction at 12 weeks for hiPSC-MSC–CPC constructs was (30.4±5.8)%, which was 2.8-fold the (11.0±6.3)% of CPC control without cell seeding (P<0.05). No significant differences in new bone area fraction were detected among hiPSC-MSCs, hUCMSCs and hBMSCs groups (P>0.1). The new bone exhibited an organized morphology which is typical of mature bone, manifested by the appearance of bone matrix with osteocytes and blood vessels, and with newly formed bone being lined by osteoblasts (Figure 4). Thus, hiPSC-MSCs and hUCMSCs may represent viable alternatives to hBMSCs and hESCs for bone regeneration. Reddy et al.104 compared MSCs from four different sources (human placenta, umbilical cord, fetal bone marrow and adipose tissue), cultured in the presence of nanosized biphasic ceramics. Placental MSCs demonstrated the best osteogenic potential based on expression of osteogenic markers and complete regeneration of bone defect in the femur of rats when seeded with nanoceramic with a Ca/P ratio of 1.58. Therefore, constructs using stem cells and nano-CaP scaffolds are highly promising for bone regeneration in vivo.
The above-mentioned studies only seeded cells onto the surfaces of preformed CaP scaffolds. This type of static cell seeding method has limitations of low seeding efficiency and minimal cell penetration into scaffold, leading to non-uniform distribution of cells.105 To address these problems, cell micro-encapsulation has been proposed as another approach to deliver cells. Alginate is commonly used for cell delivery owing to its gentle gelling ability, and free of detrimental effects to encapsulated cells. However, the degradation of alginate is undesirably slow and uncontrollable.106 Therefore, fast degradable alginate hydrogels were developed via partial oxidation, and the addition of fibrin further accelerated the degradation to as early as 4 days.107 Our recent study encapsulated hiPSC-MSCs in the fast degradable alginate-fibrin microbeads, mixed with CPC paste (which was reinforced by 10% absorbable fibers), and then transplanted into nude rats using a double cranial bone defect model. The CPC paste contained 50% of alginate-fibrin microbeads which encapsulated hiPSC-MSCs. The control group had the same CPC compositions with the same alginate-fibrin microbeads but without encapsulating cells. As shown in Figure 5, the cell-encapsulated group generated significantly more new bone than the counterpart without cell encapsulation at 3 months. The average new bone area fraction in the ‘CPC–10% fibers–50% beads with hiPSC-MSCs’ group was (38.9±18.4)%, more than twofold the (15.6±11.2)% of the control group (P<0.05) (Figure 5). In other studies, alginate microspheres and chitosan-coated alginate microspheres were used to encapsulate cells.108,109 Then the microspheres were dispersed in CPC paste for injection, one for subcutaneous implantation in nude mice,108 and the other for rabbit femoral condylar defects.109 In vivo implantation showed that cell-encapsulated groups degraded faster than the cell-free groups and exhibited higher mineralization and bone forming ability.108,109 The encapsulated cells were still detectable in the graft areas 8 weeks postoperatively.109 These in vivo results indicated that the encapsulated cells can be released from the microspheres and survived in vivo, while concomitantly creating pores in the scaffolds. These studies demonstrated that cell microencapsulation in combination with nano-CaP scaffolds is a promising tool for enhancing the bone tissue engineering efficacy.
Nanostructured CaP biomaterials and scaffolds mimic natural bone, and have high surface-to-volume ratios, improved wettability and mechanical properties, and increased protein adsorption and other desirable properties, compared to conventional counterparts. Nano-CaP biomaterials have emerged as a promising class of biomimetic and bioactive scaffolds capable of directing cell behavior and cell fate and enhancing tissue formation in vivo. In general, nano-CaP scaffolds can support stem cell attachment and proliferation and induce osteogenic differentiation, in some cases without osteogenic supplements. The influence of nano-CaP on cell alignment is less prominent than polymers and metals, due to the non-uniform distribution of the nano-CaP crystals. Nano-CaP biomaterials can achieve significantly better bone regeneration in vivo than conventional CaP biomaterials. The combination of various types of stem cells with nano-CaP scaffolds can further accelerate bone regeneration, the effect of which can be even further promoted by growth factor incorporation. Cell microencapsulation in combination with nano-CaP scaffolds is a promising tool for bone tissue engineering applications to distribute cells throughout the interior of the scaffold. Further studies are needed to compare various types of nano-CaP compositions and nanostructures side-by-side in vivo, and compare the various types of stem cells in bone regeneration efficacy. Studies should also focus on understanding the bone tissue regeneration mechanisms via nano-CaP constructs, and tailoring the nano-CaP biomaterials and stem cells types to further enhance bone regeneration in vivo for various craniofacial and orthopedic applications.
We gratefully thank Dr Laurence C. Chow, Dr Carl G. Simon, Dr David J. Mooney, Dr Hongzhi Zhou and Dr Minghui Tang for fruitful discussions. This study was supported by NIH R01 DE14190 and R21 DE22625 (HX), National Science Foundation of China 31100695 and 31328008 (LZ), 81401794 (PW), Maryland Stem Cell Research Fund and University of Maryland School of Dentistry.
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