Calcium Phosphate Flowers

Bone provides a supportive structural frame work to protect sensitive parts of the body, lends support to teeth and equips the body with strength to resist load and shocks. Skeletal tissues are also involved in physiological functions such as mineral ion balance, calcium homeostasis, and blood formation. Bone exhibits a continuous adaptation known as remodeling and maintains size, shape and structural integrity, and at the same time regulating the mineral homeostasis [1]. While remodeling, bones are resorbed and grow in relation to trophic stimuli and mechanical stress [2]. This enables osseous tissues to repair defects and fractures within a critical size. However, when healing of complicated fractures, pathological bone defects may impair and require additional treatment to aid the healing process. Bone substitute grafts and implants are the most common aids to help the repair of irreversible bone defects [3]. Autografts are one of the most preferred, gold standard techniques. However, highly invasive surgery, donor site morbidity, pain, and discomfort to patients limits the quantity of autografts. Allografts from cadavers or living donors or xenografts from different animal sources are alternative approaches but are less preferred due to complications from immunogenicity, infection, and donor availability [4].

The limitation of suitable grafts led to the emergence of synthetic graft substitutes and implants. The synthetic materials interact with host cells, recruit them and induce the body’s innate powers of organization and self-repair [5]. The success of these strategies to achieve bone repair depends on three distinct properties: osteoinductivity, osteointegration, and osteoconductivity. The studies have mainly focused on types of materials and development of graft towards mimicking these properties, in solo or in combination. In this regard, different biomaterials have been investigated to provide functional surrogates of neo-tissue formation, to be employed as grafts for implantation and for physical models for controlled studies of cell behavior [6].

In different types of bone graft substitute materials, bone filler substitutes (BFSs) stand as a distinct class for the treatment of bone defects due to trauma, bone tumor resection, osteolysis around the joint prosthesis, and other pathological conditions. BFSs are important in small size defects, especially in immuno-compromised patients, or during the unavailability of auto or allografts. These substitutes are also used as a coating material for bioactivation of inert metallic devices. In recent decades, different types of demineralized bone matrices (DBMs) and various synthetic calcium phosphate (CaP) origin bone fillers have been introduced into the market. DBMs of animal origin often cause an immunogenic reaction and slow down the healing process. The shift from natural origin materials to synthetic CaP and bone cement resulted in better osteoconductivity but poor osteoinductive properties, leakage and irregular degradation that causes several local and systemic adverse effects [7,8]. Researchers explored different ways to control the resorbability and rate of bone regeneration by mixing different phases of calcium phosphate such as hydroxyapatite and β-tricalcium phosphate, with or without other minerals and polymers. In recent studies, it has been evident that natural origin CaP displayed better bone forming capabilities over chemically synthesized CaP during in vitro and in vivo studies [9]. In our previous studies, modification of natural origin calcium resources lead to multiphasic CaP and provide better balance between bone forming properties and resorbability [10]. The present study was an attempt to achieve the optimal recipe for BFSs and address the challenges with currently available bone fillers. Here, egg shells, a common natural origin calcium resource were modified via thermochemical treatment towards potential BFSs.

The synthesis of egg shell derived bone filler substitute (ES-BFS) was based on a previously described method. Briefly, raw egg shells were cleaned with 2% acetic acid and crushed to 100?µm sized particles. The prepared powder was reacted with the orthophosphoric acid at a weight ratio of 1:3 and subsequent overnight milling followed. The prepared reaction mixture was further made into premix for wet spinning in a 2:1 ratio with 4% chitosan (700?KD, 95% de-acetylated) solution in 2% v/v aqueous acetic acid by 2?h stirring. The final slurry was extruded through a syringe in 1M di-ammonium hydrogen phosphate spinning bath. The prepared fibers were dried in a controlled humidity chamber at room temperature. Later fibers were placed in a muffle furnace for binder burnout at dwelling time of 2?h at 400?°C, and 600?°C, respectively and sintered at 800?°C for 1?h.

The X-ray diffraction (Panalytical High-Resolution XRD-I, PW 3040/60) analysis of sintered fibers showed the formation of multiple crystalline phases including hydroxyapatite, tricalcium phosphate, with mono-, di-, tri-, and tetra- phases of CaP. Fourier transform infrared (Perkin Elmer FTIR spectrophotometer) studies further confirmed the presence of phosphate. The microstructural analysis using scanning electron microscopy (SEM) (ZEISS EVO 60 scanning electron microscope) displayed a highly porous irregular surface with granular morphology with indistinguishable grain boundaries due to liquid phase reaction. Moreover, significant continuous interconnected porosity with a wide range pore sizes was observed in the order of 3–20?µm on the fiber surface which will eventually facilitate cellular attachment and migration on cell seeding. The fibers showed typical flower like stacked morphology of 30–50?µm, further needle-like flakes displayed continuous interconnected porosity extending radially from the center. The total porosity of the fiber was observed to be ∼60%, further, it is noted that the flower shaped porous network provided multi-scalar porosity ranging from ∼1.6?nm up to ∼50?µm within individual fibers. The surface porosity promotes cell adhesion and migration, and inter-fibrous porosity will be beneficial for cell ingrowth, vascularization, and further tissue remodeling. It was confirmed by seeding umbilical cord derived mesenchymal stem cells (MSCs), where ES-BFSs displayed better cell attachment and migration compared to the control (chemical synthesized CaP). The cell proliferation was also elevated compared to the control. The multiphasic nature of CaP caused low-fouling activity resulting in significantly less non-specific serum protein adsorption and ultimately facilitated cell adhesion. The multi-scalar porosity, low-fouling activity, and multiphasic CaP nature caused rapid MSCs differentiation into osteogenic lineage. The transcriptome analysis of cells on ES-BFSs showed significantly higher expression of ALP, OC, osteonectin, osteopontin, and collagen-l. Further, ES-BFSs displayed promising biocompatibility after subcutaneous implantation and ectopic bone like tissue formation.

As a conclusion, the result confirms that the flower architecture of ES-BFSs provided higher surface area associated with interconnected porosity. These properties along with multiphasic nature of CaP established it as a potential bone filler substitute.

Further reading

[1] L.J. Raggatt, N.C. Partridge
J. Biol. Chem., 285 (33) (2010), pp. 25103-25108

[2] S. Weiner, W. Traub, H.D. Wagner
J. Struct. Biol., 126 (3) (1999), pp. 241-255

[3] E.S. Place, N.D. Evans, M.M. Stevens
Nat. Mater., 8 (6) (2009), pp. 457-470

[4] S. Ohba, F. Yano, U. Chung
IBMS Bonekey, 6 (11) (2009), pp. 405-419

[5] L. Meinel, et al.
Ann. Biomed. Eng., 32 (1) (2004), pp. 112-122

[6] S. Sell
Stem Cells Handbook
(1st ed.), Totowa, NJ (2004)

[7] I. Tsukamoto, M. Akagi
Acta Med. Okayama, 71 (1) (2017), pp. 19-24

[8] Q. Wang, et al.
Mater. Today, 19 (8) (2016), pp. 451-463

[9] S.-W. Lee, et al.
Oral Surg. Oral Med. Oral Pathol. Oral Radiol., 113 (3) (2012), pp. 348-355

[10] P. Dadhich, et al.
ACS Appl. Mater. Interfaces, 8 (19) (2016), pp. 11910-11924

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DOI: 10.1016/j.mattod.2017.10.009