Biomimetic Mineralization of Biomaterials Using Simulated Body Fluids for Bone Tissue Engineering and Regenerative Medicine

Abstract

Development of synthetic biomaterials imbued with inorganic and organic characteristics of natural bone that are capable of promoting effective bone tissue regeneration is an ongoing goal of regenerative medicine. Calcium phosphate (CaP) has been predominantly utilized to mimic the inorganic components of bone, such as calcium hydroxyapatite, due to its intrinsic bioactivity and osteoconductivity. CaP-based materials can be further engineered to promote osteoinductivity through the incorporation of osteogenic biomolecules. In this study, we briefly describe the microstructure and the process of natural bone mineralization and introduce various methods for coating CaP onto biomaterial surfaces. In particular, we summarize the advantages and current progress of biomimetic surface-mineralizing processes using simulated body fluids for coating bone-like carbonated apatite onto various material surfaces such as metals, ceramics, and polymers. The osteoinductive effects of integrating biomolecules such as proteins, growth factors, and genes into the mineral coatings are also discussed.

Introduction

Musculoskeletal disorders and diseases cost over $180 billion annually in the United States.¹ Demand for bone grafts and the financial burden are expected to rapidly increase due to increasing life expectancy.^2,3 Autograft bone is currently the clinical gold standard for treating critical size bone defects. Bone autografts, however, have the disadvantages of generating a second surgical site, donor-site morbidity, and diminished patient quality of life due to surgical burden.^4–6 Allograft bone and xenograft bone are alternative options to autograft bone, but these run the risk of transmission of infection, immune rejection, toxicity associated with sterilization, and being inefficient at osteoinduction.^4,5,7–9 Therefore, the field of tissue engineering and regenerative medicine has been focused on alternative ways of regenerating healthy tissue to replace diseased or damaged bone tissue.

Desirable characteristics of biomaterials for bone tissue engineering are as follows: possessing a bioactive surface^10–12; having the capacity to promote new bone formation from the surrounding established bone (osteoconductivity)^13,14; and having the ability to induce osteoblastic differentiation (osteoinductivity).^13,14 To achieve these properties, researchers have focused on developing scaffolds using a multitude of materials, including natural products, synthetic polymers, and metals that offer ideal properties for tissue engineering. However, these products still fall short of the gold standard: autografts. To improve the bone regeneration properties of scaffolds, researchers have coated them with various forms of apatite, which mimic the natural bone surface thus providing an ideal environment for osteogenesis and increase the structural stability of the scaffold. A promising approach to surface coating materials is using simulated body fluids (SBFs). The high concentration of calcium and phosphate in these fluids promotes the formation of calcium phosphate (CaP) crystalline structures similar to the apatite found in native bone. In this study, the use of SBFs as a biomimetic technique will be discussed through investigation of SBF analytical use, applicable substrates, and biomolecule incorporation.

Bone is a complex tissue that consists of an inorganic phase intimately embedded into an organic extracellular matrix (ECM). The mass of dehydrated bone is ∼70% inorganic and 30% organic.¹⁵ The inorganic phase of bone contributes to the structural support of the skeletal system. Bone mineral comprises mainly of carbonated hydroxyapatite (carbonated apatite)¹⁶ and differs from hydroxyapatite in that it is a nonstoichiometric apatite with a Ca/P ratio that may range from 1.50 to 1.90 depending on age, gender, bone site, and pathophysiological conditions. This nonstoichiometric chemical composition of bone mineral is mainly due to the presence of ionic substitutions, such as CO₃²⁻ and HPO₄²⁻ that may be substituted for PO₄³⁻, while Na⁺, Mg ²⁺, and K²⁺ may replace Ca²⁺ of hydroxyapatite.^17,18 It is anticipated that the poorly crystalline (i.e., amorphous) property, in conjunction with nonstoichiometric chemistry, of carbonated apatite contributes to it possessing a higher solubility than hydroxyapatite.^19–21 The organic ECM is predominantly composed of collagens, type I collagen in particular. In addition, noncollagenous proteins known as small integrin-binding ligand N-linked glycoproteins (SIBLING proteins) comprise 10–15% of total bone protein content.²²

Bone mineralization occurs during development, remodeling of existing bone, and fracture repair.^15,23 To form new bone, osteoblasts are recruited to the area through a variety of factors such as transforming growth factor-β and bone morphogenetic proteins (BMPs). Then, the osteoblasts begin to deposit a new collagenous matrix for mineralization to occur.^24,25 The osteoblasts then accumulate Ca²⁺ and PO₄³⁻ within polarized matrix vesicles promoted by phosphatases, calcium binding proteins, and potential mitochondria vesicles.²⁶ The matrix vesicles are then released from osteoblasts and continue to concentrate Ca²⁺ and PO₄³⁻.²⁷ Once precipitation occurs, the matrix vesicles release hydroxyapatite nanocrystals and amorphous calcium phosphate into the local environment.¹⁵ The collagen framework, now coated with highly acidic fibrils such as SIBLlNG proteins, provides an anchoring point for the nanocrystals.^16,24 The attached crystals and charged regions of the collagen act as nucleation sites for crystal growth by converting the high levels of Ca²⁺, PO₄³⁻, and amorphous calcium phosphate into ordered carbonated apatite (Fig. 1).^26,28 Over time, osteoblasts become embedded within the bone mineral and collagen matrix, and differentiate to osteocytes or undergo apoptosis.¹⁵

FIG. 1.

Natural osteoblast-mediated bone mineralization. (1 and 2) Osteoblast matrix vesicles accumulate calcium and phosphate ions (transitioning from yellow to white) from the cytosol and mitochondria, and are released toward the newly formed collagen matrix (red lines). (3) The released vesicles continue to concentrate calcium and phosphate ions until precipitation occurs, drawing from the ion-rich environment. (4) The newly formed apatite crystals (white crosses) are released into the environment, (5) providing nucleation sites for continued apatite growth. Adapted from Mescher 2013, The McGraw-Hill Companies, Inc.²⁸ Color images available online at www.liebertpub.com/tea

Approaches for Mineralizing Biomaterials

Biomaterials coated with CaP have been widely used for bone regeneration due to their excellent intrinsic bioactivity and osteoconductivity. CaP-based coatings can be further engineered to incorporate biomolecules that promote osteoinductivity. CaP coatings were first investigated in the early 1980s to treat the surface of titanium (Ti) metal implants so as to enhance the bonding ability of the implant to the bone. Since then, various methods have been developed to provide bioactivity to nonbioactive materials using various coating techniques such as thermal spraying,^29–33 sputter coating,^34–36 sol–gel deposition,^37–39 hot isostatic pressing,^40,41 and dip coating.⁴² Each of these methods has advantages and disadvantages (Table 1).⁴³

Table 1.

Calcium Phosphate Coating Techniques for Biomaterials

Method	Advantages	Disadvantages	Process conditions	Coating thickness	References
Thermal spraying	Widely commercialized with low processing cost; high coating rate	Biomolecules cannot be incorporated due to high processing temperature; large minimum thickness for uniform coating; low adhesive strength; risk of delamination	High processing temperature (>8,000°C)	30–200 μm	Huang et al.29; De Groot et al.30; Yang et al.31; Li et al.32; Gross et al.33
Sputter coating	Can form thin and uniform coating; high adhesive strength	Long processing time; expensive; hard to control Ca/P ratio when processed by RF magnetic sputtering;	Low processing temperature	<3 μm	Surmenev34; Ding35; Wolke et al.36
Sol–gel deposition	Can uniformly form thin coatings on complex substrates	Expensive raw material; some processes require high sintering process	Low processing temperature	<1 μm	Nguyen et al.37; Liu et al.38; Manso et al.39
Hot isostatic pressing	Can produce dense coatings	Cannot coat complex substrates; expensive; high temperature and high pressure involved	High processing temperature and pressure	0.2–2.0 mm	Li et al.40; Onoki and Hashida41
Dip coating	Inexpensive; short processing time; can coat complex substrates	Needs sintering process; thermal expansion mismatch	High sintering temperature	0.05–0.5 mm	Mavis and Taş42
Biomimetic coating with SBF solution	Can form bone-like apatite; mild processing conditions allow incorporation of biomolecules; can coat complex substrates	Long processing time; ionic concentration and pH of SBF need to be maintained by replenishment; uniform coating under static conditions is limited by certain thicknesses	Biocompatible; pH close to or slightly lower than 7.4; atmospheric pressure; incubation at 37°C	<30 μm	Shin et al.45; Kohn et al.46; Liu et al.49; Habibovic et al.50; Kokubo and Yamaguchi52; Choi and Murphy93

Adapted from Ong et al. (2009), with permission from Springer Science + Business Media.⁴³

CaP, calcium phosphate; RF, radio frequency; SBF, simulated body fluid.

Thermal spraying is one of the most successful and widely commercialized techniques used for CaP coating.^29,30 The technique involves feeding the coating material into a plasma jet, where the sample is heated to >8,000°C and then propelled toward the desired surface (Fig. 2).⁴⁴ The high processing temperature may limit the selection of underlying substrate materials, and be problematic when incorporation of heat labile biological molecules is desired. It also requires a relatively large thickness (30–200 μm) to achieve uniform coating³¹ and is therefore not ideal for small-sized materials or intricate structures. In addition, this technique has the disadvantage of low adhesive strength and risk of delamination.

FIG. 2.

Plasma spray coating. A plasma jet is created when the plasma gas passes through the electric field generated by the anode and cathode. Then, the coating powder is injected into the plasma jet, which rapidly propels the material onto the substrate. Adapted from Bosco et al. 2012, MDPI.⁴⁴ Color images available online at www.liebertpub.com/tea

Radio frequency (RF) magnetron sputtering involves an RF generator, a magnetron, and an ionizable gas.³⁴ The generator and magnetron efficiently convert the gas into the plasma, which is directed to bombard the coating material. The coating material is then ejected toward the desired substrate (Fig. 3).⁴⁴ This method provides a great deal of control over the coating material by fabricating thin (<1 μm), uniform CaP coatings with high adhesive strength. However, RF magnetron sputtering is a time-intensive and high-cost method that only coats the visible surface of the substrate.³⁴

FIG. 3.

RF magnetron sputter coating. The magnetic field forces the plasma close to the cathode, and the alternating current of the RF generator prevents charge build up in concentrated areas. The high-energy ions of the plasma bombard the coating material (target) ejecting the material toward the substrate producing a thin uniform coat. Adapted from Bosco et al. 2012, MDPI.⁴⁴ RF, radio frequency. Color images available online at www.liebertpub.com/tea

Of all of the aforementioned methods, sol–gel deposition is the only one that can achieve uniform coating throughout a porous matrix.³⁷ The sol–gel method involves forming a solution (“sol”) containing the calcium and phosphate to be coated, followed by dipping the substrate into the solution and allowing it to dry to form a viscous gel-like layer.³⁸ The gel-like coating can be calcinated to form a hardened layer of apatite on the substrate (Fig. 4). Since the sol is highly fluid, the sol–gel deposition technique is able to coat the interior of a porous substrate. However, high processing costs and expensive raw materials for this method are often prohibitive.³¹

FIG. 4.

Sol–gel coating. The precursor solution undergoes polymerization resulting in a gel-like solution (Sol). Then, the substrate is dipped into the Sol, removed, and allowed to dry to a thick film. Xerogel film is sintered using a drying oven. This leads to polycondensation and enhances the mechanical properties of the final dense film. Color images available online at www.liebertpub.com/tea

Some techniques, such as thermal spraying, dynamic mixing method, and isostatic pressing, require high processing temperatures and are therefore not amenable to polymers with relatively low melting temperatures. Also, many techniques, such as plasma spraying, sputter coating, pulsed laser deposition, and dynamic mixing method, are limited to one-sided (“line of sight”) coating, as their coating processes are unidirectional. This is not desirable for coating materials with three‐dimensional (3D) complex structures.

Biomimetic Mineralization by SBFs

A promising alternative CaP coating method is mineralization of material surfaces using a supersaturated solution known as SBFs. SBFs comprised ions at similar concentrations to those found in blood plasma. This coating technique is performed under biological conditions in terms of temperature, pressure, and pH, forming carbonated apatite on a substrate, which is similar in chemical composition and material properties (crystallinity and dissolution rate) to bone mineral (Fig. 5). Mineral formation using this biomimetic process is governed by both the surface characteristics of the materials and the immersion parameters, such as the composition of the SBF, ionic strength, pH, temperature, and immersion time.^45–50 Since the CaP layers are coated using aqueous SBF solutions, surfaces of highly complex structure such as 3D interconnective porous scaffolds can be uniformly coated, unlike other conventional “line-of-sight” CaP coating techniques described above. The coating conditions of this technique, such as pH and temperature, are similar to those of body fluid, which allows for a wide range of candidate materials to be coated with CaP. Furthermore, these biocompatible conditions enable the potential use of biomolecules sensitive to pH and temperature, such as proteins, growth factors, and genes.

FIG. 5.

SBF-mediated mineralization. (1) The high concentrations of calcium and phosphate ions contained in the SBF begin to form prenucleation crystals. (2) The amorphous solids are attracted to the polar surface groups of the substrate. (3 and 4) Apatite crystals are deposited. (5) The formed apatite is a nucleation site allowing for continued crystal growth. SBF, simulated body fluid. Color images available online at www.liebertpub.com/tea

Kokubo et al. introduced the concept of biomimetic mineralization using SBFs in 1990.⁵¹ Glass-ceramic A-W (apatite-wollastonite) was soaked in various aqueous solutions possessing similar ionic concentrations and pH levels to human blood plasma. After incubation for 7–30 days, they reported that an apatite phase had formed on the glass-ceramic surface. Since this initial report, the use of SBF to form bone-like apatite has been extended to various types of materials such as metals, ceramics, and biodegradable polymers. Over the past three decades, SBF has been widely used and developed for the following purposes: (1) bioactivity assessment for biomaterials, (2) surface coating of biomaterials to improve osteoconductivity, and (3) incorporation of biomolecules into mineral coatings.

Bioactivity assessment

From the 1970s, several materials have been reported as bioactive through their ability to integrate with host bone tissue via apatite formed on the interface between the material and the bone subsequent to implantation in vivo. From these observations, it has been suggested that the apatite-forming ability in vivo can be pretested in vitro using solutions that simulate body fluid. Numerous biomaterials such as metals,⁵² natural polymers,^53–55 synthetic polymers,^56,57 and organic/inorganic composite materials^58,59 have been tested for their potential apatite-forming ability in the presence of SBF solutions.^{10,11,52–61} Various methods for testing the bioactivity in vitro using SBF solutions have been standardized.⁶² A recently published review article reported on how in vitro apatite-forming ability in the presence of SBF could often successfully predict actual bioactivity of biomaterials in vivo using animal models.⁶⁰ SBFs also have been used to test other material properties such as polymer biodegradation,^63,64 and wear or corrosion behavior of metals.^65,66

Surface coating of biomaterials to promote osteoconductivity

The apatite coating technique using immersion in SBFs can be applied to various types of materials, including metals,⁵² ceramics,^51,67,68 polymers,^45,69–72 and organic/inorganic composite materials.⁷³ To achieve successful apatite coating, the surfaces of the materials need to be modified to be functionally activated.

Metals

Ti metal and its alloys are among the most commonly used metals for dental implants and bone substitutions. Various kinds of surface treatments have been attempted to confer bioactivity or apatite-forming ability on Ti metal and its alloys. Heat treatment with NaOH solution can form a sodium hydrogen titanate (Na_xH_2-xTi_yO_2y+1; 0<x<2) layer with functional groups of Na⁺ and O²⁻ on the surface. The treated Ti metal formed bone-like apatite on its surface after immersion in SBF, while the nontreated Ti metal did not.⁷⁴ Surface treatment methods have been modified to enhance osteoconductivity of Ti metal, including NaOH/CaCl₂/heat treatments, H₂SO₄/HCl/heat treatments, and NaOH/acid/heat treatments.

Metal implants coated with bone-like mineral apatite using SBFs also resulted in enhanced osteoconductivity in vivo compared with noncoated metal implants. Significantly greater bonding strength of the interface between the implant and the bone was obtained with bone-like mineral-coated Ti alloy implants in goat femurs, compared to noncoated implant groups.^19,75 This enhanced bonding strength can be attributed to the precoated bone-like mineral layer that promotes new bone deposition onto the osteoconductive surfaces.

Ceramics

Ceramics are another class of biomaterials that have been extensively studied because of their superior bioactivity and potential application for dental or skeletal tissue repair.^{51,68,76–79} Ceramics are well suited for SBF treatment because, once immersed in SBF, ceramics can release ions such as calcium and silica. These released ions contribute to nucleation and subsequent surface mineralization, thus forming bone-like mineral apatite. Many types of ceramics, such as Bioglass 45S5, glass-ceramic A-W, and glasses in the Na₂O-CaO-B₂O₃-Al₂O₃-SiO₂-P₂O₅ system, were able to become bioactive by forming mineral layers on their surfaces in the presence of SBF. These ceramics were confirmed to bind to the living bone through newly formed CaP layers at the interface between the implant and the bone when they were implanted in vivo.¹⁰

Polymers

Although metals and ceramics serve as adequate materials for bone and dental implants due to their potential bioactivity, the nondegradability of these materials limits their application for bone tissue engineering. Adjustable biodegradability of the scaffold is essential for bone tissue engineering. The scaffold needs to persist for sufficient time to allow new bone tissue formation to occur. Then, as the scaffold degrades, it will be substituted with the regenerated bone. Therefore, natural and synthetic biodegradable polymers have been widely used as scaffold materials for tissue engineering due to their advantages such as controllable biodegradation and tunable scaffold properties.

Natural polymer-based scaffolds that are nontoxic and bioactive can provide cells with a biocompatible microenvironment. Natural polymers used as scaffold materials can be divided into proteins (collagen,^76,80,81 silk,⁸² and gelatin^80,83,84) or carbohydrates (cellulose^85–87 and chitin^54,55,88,89). Multiple natural polymers have been incubated in SBF to obtain biomimetic mineral properties and then used in organic/inorganic scaffolds for the purposes of promoting tissue regeneration.

Biomimetic surface mineralization by SBF immersion has also utilized multiple types of synthetic polymers such as poly(lactide-co-glycolide) (PLGA),^{45,47,49,69,72,90–98} poly-L-lactide,^98,99 poly(2-hydroxyethylmethacrylate), poly(ɛ-caprolactone),^99,100 and polyhydroxyalkanoate.⁵⁶ Compared to natural polymers, synthetic polymers have found widespread application as scaffold candidate materials, as their mechanical and chemical properties can be specifically controlled.^71,101,102

Incorporation of Biomolecules by Biomimetic Mineralization

Although biomimetic mineral coatings provide the underlying scaffold with osteoconductivity, they do not directly confer osteoinductivity. To overcome this limitation, current research has been focused on integrating drugs or biological molecules such as proteins and genes into the mineral coatings (Fig. 6). In this study, some examples of drugs, proteins, and genes incorporated into the CaP coatings will be introduced, and their therapeutic benefits will be assessed.

FIG. 6.

Incorporation of biomolecules by biomimetic mineralization. To further functionalize the coating created by SBFs, proteins, drugs, or genes can be incorporated into the apatite coatings. In this study, the ions present in SBF are displayed alongside the different therapeutic molecules that can be incorporated into the apatite coating of the substrate. BMP, bone morphogenetic protein; FGF, fibroblast growth factor; IGF, insulin-like growth factor. Color images available online at www.liebertpub.com/tea

Drugs

Currently, there are a multitude of drug molecules being investigated for their potential incorporation into the CaP coatings to reduce inflammation and enhance osteogenesis. Incorporation of antibiotics into the CaP layer can prevent postoperative infection at the surgical site promoting favorable osteointegration of dental/skeletal implants and bone substituting materials.¹⁰³ This is highly desirable, because of the prevalence of peri-implantitis. Unfortunately, conventional CaP coating techniques, such as plasma spraying, and isostatic pressing involving nonphysiological processing conditions, such as high temperature and high pressure, do not allow incorporation of drugs into the CaP coatings. Various antibiotics were successfully incorporated into CaP coatings on Ti implants using SBFs.¹⁰³ The Ti surface was initially coated with a thin layer of amorphous carbonated apatite by immersing the metal in a supersaturated SBF solution, and then antibiotics were coprecipitated. Loading ability, release kinetics, and efficacy of the antibiotics were evaluated. Antibiotics containing carboxyl groups, such as cephalothin, carbenicillin, and cefamandole, possessed higher binding affinities and slower releasing kinetics, suggesting that the chemical structure of the antibiotics determined their binding/chelating affinity to calcium-rich mineral coatings.¹⁰³

Bisphosphonates (BPs) are primary agents for treating osteoporosis. However, current publications have demonstrated that systemic delivery of BPs results in inefficient dose delivery to the target site and causes toxic side effects, such as gastric ulcers and BP-related osteonecrosis of the jaw. All BPs have the same backbone (P-C-P),¹⁰⁴ which provides them with a high calcium-binding affinity and enables the incorporation of BPs into CaP layers through SBF-mediated coating. For example, alendronate sodium (AS), an approved BP, was successfully incorporated into calcium-deficient hydroxyapatite coatings on Ti alloys using a biomimetic coating process.¹⁰⁵ The release profile of AS was controllable through modification of the SBF-mediated incorporation. These results suggest that drug incorporation in the mineral coating through SBF mediation can be optimized to provide a localized, long-acting administration to achieve a therapeutic dose.

Proteins and growth factors

Proteins can be adsorbed onto mineral surfaces forming organic/inorganic hybrids. Once bone-like mineral coatings are formed on substrate materials, proteins are then adsorbed on the mineral surface via electrostatic interactions between proteins and the mineral apatite. As this interaction occurs after the surface is established, proteins do not integrate into the mineral structure nor alter mineral formation. Proteins can also be incorporated within the mineral coating by coprecipitation in SBF solution. These different methods (surface adsorption vs. coprecipitation within mineral structure) affect the release kinetics of the incorporated proteins. The surface-adsorbed proteins demonstrate a burst release profile, whereas the coprecipitated proteins show a more controlled and sustained release profile, for the proteins are physically incorporated with the mineral layers.^106–110

As a proof of concept, osteogenic growth factors were combined with mineral coatings to further the osteogenic capabilities of the biomaterial. After CaP layers were formed using conventional coating methods, BMP-2 was superficially deposited on the outer surface of these mineral coatings, through either adsorption¹⁰⁶ or chemical surface treatment.¹⁰⁷ Both of these superficially adsorbed growth factors were released with a pattern of initial burst release (higher release rate), however, this release profile is not the ideal delivery kinetics for biological outcomes.

Sustained release of proteins or growth factors allows for long-term delivery within the therapeutic range and can be achieved by incorporating proteins or growth factors into the mineral coating. This concept of controlling release profile has been applied to the biomimetic mineralization process. Coprecipitation incorporates biomolecules, such as proteins, growth factors, enzymes, and drugs, into the bone-like mineral coatings. BMP-2-incorporated mineral coatings fabricated by a biomimetic coprecipitation technique in SBF have been compared to mineral coatings with superficially adsorbed BMP-2.^108,109 A pharmacologically favorable low dose of BMP-2 was gradually released from the groups containing BMP-2 incorporated by coprecipitation, whereas a burst release of BMP-2 was observed from the groups with superficially adsorbed BMP-2. With the same amount of BMP-2 loaded by the two different methods, a more sustained osteogenic response was observed in the groups where BMP-2 was incorporated by coprecipitation.¹¹⁰ In another study, the delivery mode and efficacy of BMP-2 were tested using Ti-alloy (Ti6A14V) discs implanted subcutaneously in the dorsal region of rats for up to 5 weeks. Significantly improved bone volume and density of the regenerated bone were observed in the groups that provided sustained delivery of BMP-2.^49,108,109 Furthermore, the release kinetics of insulin-like growth factor-1 (IGF-1) was linear with a sustained profile, when IGF-1 was incorporated within mineral coatings by coprecipitation.¹¹¹

Gene therapy

Gene therapy has attracted scientific interest due to its advantages over protein-based growth factor delivery. Current problems with protein-based delivery include the following: (1) continuous administration of protein-based growth factors is required for biological outcomes, (2) optimized spatiotemporal delivery is challenging and, if not achieved, cost and efficacy are prohibitive, and (3) multiple doses are required due to the short half-life of protein-based growth factors.^112,113

Developing gene delivery carriers has been extensively studied,^{92,96,113–123} and biomimetic mineralization using SBF has been investigated as a means to synthesize a nonviral gene delivery agent. The physiological conditions (temperature, pH, ionic composition of SBF) used during the biomimetic mineralization process allow incorporation of genetic material with low risk of denaturing the DNA. The negative charge on the DNA provides a nucleation site for the high concentration of calcium ions to precipitate forming CaP/DNA complexes in the SBF. Therefore, precipitation of DNA/calcium-containing composites has been successfully formulated by using SBFs or modified SBFs for nonviral gene delivery.^115,121,124 Prefabricated coprecipitates of model DNA (lambda DNA) encapsulated in CaP (DNA/CaP) were adsorbed onto 2D PLGA plates and 3D interconnective porous PLGA scaffolds. Although human bone cell line (SaOS-2) was successfully transfected onto 2D plates and 3D scaffolds, more than 95% of the initially adsorbed DNA/CaP was released within 2 days.¹²⁴

Naked plasmid DNA (pDsRed pDNA) was superficially adsorbed onto mineral-coated PLGA film.⁹⁴ The release kinetics of pDNA was modulated by both the intrinsic properties of the minerals formed in different SBFs and the extrinsic conditions such as pH and ionic composition of the testing solutions. The same group also demonstrated pDNA (pMetLuc and pEGFP-N1)-Lipofectamine complexes adsorbed to mineral-coated tissue culture polystyrene for testing optimized surface-mediated transfection by adjusting the carbonate content in the SBF solutions.⁹²

As with proteins, DNA can be associated with mineral coatings of prospective implants either through superficial binding of the DNA to the mineral surface by adsorption or by incorporation within mineral structure by coprecipitation. To compare surface adsorption and coprecipitation of plasmid DNA during biomimetic mineralization in terms of transfection efficiencies, plasmid DNA encoding for the β-gal gene was complexed with Lipofectamine, and integrated with bone-like mineral coatings using the two different methods. DNA-lipoplex stability was retained in both methods, but coprecipitated DNA-lipoplexes induced higher transfection efficiencies compared to adsorbed DNA-lipoplexes.⁹⁶

Limitations of Biomimetic Mineralization Using SBFs

Although biomimetic mineralization can be widely applied to enhance the tissue regenerative capacity of implanted materials, there are still a number of drawbacks and hurdles to overcome. The formation of a continuous layer of mineral coating substrate materials is a process that takes longer than other conventional CaP coating methods. A few approaches have been proposed to accelerate the biomimetic mineral coating process.^125,126 For instance, substrate surfaces can be functionalized before immersion in SBFs. The surfaces of substrate materials, such as PLGA, can be functionalized by treating them with NaOH solution resulting in the exposure of more hydroxyl functional groups on the PLGA surface and therefore increasing the capacity to bind Ca²⁺ ions in SBFs.^45,92,96 Another approach for accelerating the mineralization process is to adjust concentrations of selected ions, typically calcium and phosphorus, in SBF solutions.^48,127

Manufacturing challenges arise when using biomimetic mineral coating techniques to coat 3D interconnective porous scaffolds due to the fact that biomimetic coating under static conditions cannot uniformly coat the inner surfaces of 3D scaffolds. The nonuniform coating is particularly problematic when the scaffold is large and the pore size is small. To overcome this issue, a dynamic perfusion technique can be applied to achieve uniform coating throughout the thickness of the scaffolds.¹²⁸

A disadvantage of using the coprecipitation technique is the low efficiency of incorporation of biomolecules into mineral coatings. Only a small proportion of biomolecules in SBF can be entrapped within the mineral deposited onto the underlying substrate material.

Conclusions and Future Directions

Over the last three decades, the process of regenerating bone using SBF has made significant advances. However, the lack of SBF use in clinical practice indicates the need for further advances to overcome the translational challenges. Valuable studies investigating the role CaP in bone tissue engineering demonstrate the importance of this inorganic phase with regard to bioactivity, osteoconduction, and osteoinduction. In this review, we provided evidence that biomimetic mineralization is a promising approach to generating CaP coatings on supportive structures made from ceramics, metals, or polymers. Coating these materials with bone-like mineral layers not only increases load-bearing mechanical strength but also provides bioactivity, osteoconductivity, and, once incorporated with relevant biomolecules, osteoinductivity. In addition, it was highlighted that coprecipitation of proteins/growth factors and DNA within the mineral coatings can provide sustained delivery of these biomolecules, thereby enhancing bone tissue regeneration.

Potential future directions for biomimetic mineralization using SBF include surface mineralization of complex scaffolds fabricated by 3D bioprinting technology, improvement of SBF coating rate through surface preparation, and optimization of apatite coatings containing relevant therapeutic agents for specific diseases.

Acknowledgments

We acknowledge the financial support from the 2017 Martin “Bud” Schulman Postdoctoral Fellowship Award by the American Association of Orthodontists Foundation and the Lyle and Sharon Bighley Endowed Chair of Pharmaceutical Sciences.

Disclosure Statement

No competing financial interests exist.