Silk fibroin/hydroxyapatite scaffold: a highly compatible material for bone regeneration

Figures & data

Figure 1. Publication frequency of SF and applications of SF as a biomaterial based on Scopus database: (a) SF-related publications, (b) SF used for tissue engineering, (c) SF used for bone tissue engineering, (d) SF/HA composites, and (e) to (h) remarkable studies related to parts (a) to (d) [7]

Figure 2. Structure of silk fibroin [18]

Table 1. Structural difference between cocoon’s and spider’s silks

No.	Features	Silk of cocoons	Silk of spider
1	Size	600-1500 m from single silkworm	137 m from ampullate gland of spider and 12 m from spider web
2	Major amino acids	Glycine, alanine, serine	Glycine, alanine, glutamic acid, proline, arginine
3	β-Sheet structure	Yes	Yes
4	Components of crystalline domain	Glx (x = alanine, serine, threonine, valine)	(Alanine or glycine) + GPGX (X = proline,glutamine)
5	Components of less crystalline domain	Fibroin heavy chain made up of 25 identical amino acids	GGX glycine helix

Figure 3. Mechanical strength comparison of silks obtained from the silkworm Bombyx mori drawn at different speeds [29]

Table 2. Dominance of cocoon silk over spider silk

NO.	Properties	Bombyx mori SF without sericin	Crosslinked collagen	Polylactic acid
1	Modulus (GPa)	15–17 [30]	0.0018–0.046 [31]	1.2–3.0 [32]
2	Ultimate tensile strength (MPa)	610–690 [30]	47–72 [31]	28-50 [32]
3	% strain at break	4–16 [30]	12–16 [31]	2-16 [32]
4	Water contact angle (°)	—	84.2 ± 0.8° [33]	—

Table 3. Comparison of SF with other biomaterials

No.	Points of dominance	Cocoon silk	Spider silk [28]
1	Size	600–1500 m from single silkworm	137 m from ampullate gland of spider and 12 m from spider web
2	Nature of structure	Homogeneous	Heterogeneous
3	Domestication	High	Less
4	Productivity	High	Less
5	Processing	Easy	Difficult

Figure 4. Schematic representation of extraction of SF from cocoons [41]

Figure 5. Schematic representation of materials fabricated from silk fibroin: 4 days are required for complete extraction of silk fibroin, and different materials can be obtained at different intervals [41]

Table 4. Clinical applications of silk scaffolds

Applications	Type of tissue	Scaffold format
Tissue engineering	Bones	Sponges (both HFIP and aqueous)
		Fibres
	Cartilage	Sponges (both types)
		Fibres
	Soft tissue	Sponges
	Vascular tissues	Fibres
	Cervical tissues	Aqueous sponges
	Cornea	Films
	Skin	Fibres
Drug delivery	Drug delivery	Microspheres
	Growth factor delivery	Microspheres
	Small molecules	Microspheres
Implant material	Anterior cruciate ligament (ACL)	Fibres
	Femur defects	HFIP sponges
	Mandibular defects	Aqueous sponges
Disease modelling	Breast cancer	HFIP sponges
	Autosomal dominant polycystic kidney disease	Aqueous sponges

Figure 6. (a) atomic structure of HA and (b) its projection along c-axis [60]

Table 5. Characteristics of hydroxyapatite obtained from mammalian sources

No.	Source(animal+ extraction method)	Calcium/phosphorus ratio	Shape	Calcination (T/°C)
1	Bovine, sheep and hen femur bone +sheep skull.	1.46–2.01	Irregular	600-1100 [65]
2	Defatted pork bone pulp	—	—	650-950 [66]
3	Cow bone by (a) hydrothermal hydrolysis (b) subcritical H2O extraction (c) thermal decomposition	1.52–1.9	Nanorods	[67] 250 275 750
4	Cow bone	1.9	—	1000 [68]
5	Defatted bovine bone	1.7	Needle shaped	800 [69]
6	Cow bone through thermal and mechanochemical way	—	Spheroidal and polygonal	800 [70]
7	Bovine bone through transferred arc plasma	1.93	—	[71]
8	Human, pig and porcine bones	—	—	600-1200 [72]
9	Bovine bones by ball milling	—	Spherical	800-1100 [73]
10	Cortical femoral bovine bone	—	Equiaxial	900 [74]
11	NaOH-treated bovine bone	>3	Interconnected with pores	900 [75]
12	Cow femur bone	Almost 1.6	Round	[76]

Table 6. Properties of HA obtained from fish sources

No	Source	Ca/P ratio	Shape	Calcination (°C)
1	Big eye tuna bone	1.76	Rod like	900 [77]
2	Pseudoplatystoma corruscans, Paulicea bones	1.64	Rod like	900 [78]
3	Sepia officinalis bones	1.64	Rod	900 [79]
4	Thunnus thynnus and Xiphias gladius sword bones	~1.9	Rod	600 and 950 [80]
5	Oreochromis niloticus scales	1.7	Hexagonal	950 [81]
6	Nile tilapia scales	1.8	Occasionally circular	950 [82]
7	Gadus morhua bone	1.5	Needle like	900-1200 [83]

Table 7. Properties of HA from biogenic sources

No.	Source	Ca/P ratio	Particle size (nm)	Morphology	Secondary phase	Calcination (T/°C)
1	Egg shell	1.65	—	—	β-TCP	900 [91]
2	Coral shells	—	—	—	—	— [91]
3	HCl+DAHP solution treated Egg shells	1.67	50	Rectangular	—	[92]
4	Hen Egg shells	>1.67	18	Spherulite	—	800-1200 [93]
5	Chicken Egg shell	—	~35	Prolate spheroidal	—	700 [94]
6	Hen Egg shell	1.63	—	—	—	[95]
7	Egg shells treated with *DAHP+EDTA		78	Flower like	—	[116]
8	Egg shell treated by precipitation technique	1.67	~35	Globule like	—	900 [96]
9	Oyster shell		—	Rod like	—	1000 [97]
10	Ground waste egg shell			Flower like	—	— [98]
11	Fruits and egg shell waste	1.57–1.77	12-49	Needle like and rod like	—	— [99]
12	Egg shell waste	2.20	—	Spheroidal	β-TCP	1100 [105]
13	Mussel shell	1.61	—	—	—	— [100]
14	Egg shell	—	—	—	—	600 [101]
15	Egg shell	—	15 k-35 k	Flower like	—	— [102]
16	Sea urchin shell	—	—	Rod like	—	900 [103]
17	Egg shell	–	60	Whiskers	CaHPO4	700 [104]

* DAHP = ‘2,4-diamino-6-hydroxypyrimidine and EDTA = ethylenediaminetetraacetic acid’

Figure 7. Typical transmission electron microscopy images of mesoporous HA calcined at 600 ° C: (a) 100 nm scale and (b) 20 nm scale [109]

Figure 8. FE-SEM images of calcium phosphate nano-particles. (a–c) Nano-particles synthesized at 40 C: (a) 10% F127; (b) 40% F127; and (c) 80% F127. (d–f) Nano-particles synthesized at 100 C: (d) 10% F127; (e) 40% F127; and (f) 80% F127. Magnification is 80,000 times [108]

Figure 9. Schematic representation of preparation of SF/HA

Table 8. HA-based biomaterials in bone regeneration

No.	Composite	Method of preparation	Advantages	Disadvantages
1	HA-Chitosan	In situ chemical synthesis, freeze drying, co-precipitation	Pore forming ability, good binding capacity, anti-bacterial and biodegradable	Mechanical properties need to be improved [122–134]
2	HA-Collagen	Supercritical fluid assisted process, cryogelation technique	Better cell attachment, proliferation and differentiation ability	Presence of charged and polar groups which critically affect the nucleation of the HA crystals on collagen membrane through chemical interaction [136–153]
3	HA-Polycaprolactone (HA/PLC)	Modified rapid-prototyping for nHA/PLC, pressure quench nHA/PLC	Good biodegradation, mechanical strength, growth of MSCs and guide their osteogenic differentiation.	Difficulty in new bone tissue binding [2,152–167]
4	HA-Polyvinyl alcohol	Freeze/thaw, spray drying	Good hydrophilicity, excellent chemical stability, useful for bone tissue engineering and articular cartilage repair.	Need further in vitro and in vivo studies [8,9,168–177]
5	HA-Poly(lactic-co-glycolic) acid	Selective laser sintering, Electrospinning	Good compressive strength and modulus of elasticity	Smaller pore size [178–182]

Figure 10. Types of polymers used in HA/polymer scaffolds

Figure 11. FE-SEM images of pure SF after three cycles of Ca-P treatment and mineralized SF/HA nanofibres: (a) pure silk nanofibres and (b–d) SF/HA nanofibres with different magnification [40]

Figure 12. FTIR spectra of SF/HA porous scaffolds with different molar% contents of nHA: (a) 0%, (b) 10%, (c) 30%, (d) 60%, (e) 70%, and (f) 100% [184]

Figure 13. Elemental analysis of calcium (Ca), phosphorus (P), with oxygen (O) and nitrogen (N) in (a–d) Increase in SF/HA concentration is confirmed via EDS mapping of Ca (e–g) [185]

Figure 14. XRD patterns of different scaffolds of SF/HA composites with (a) HA0, (b) HA10, (c) HA30,(d) HA60, and (e) HA70 [184].

2θ (degree)

Figure 15. (a) Water contact angle of SF/HA scaffolds with different HA contents (p < 0.05) and (b) photographs of water droplets on SF/HA scaffolds [185]

Figure 16. Thermogravimetric curves of nHA, pure SF and SF/HA (molar ratio = 8:2) [40]

Figure 17. SEM images of porous SF/HA scaffolds with different molar contents of nHA: (a) 10% (b) 0% (c) 10% (d) 30% (e) 60% and (f) 70% [184]

Figure 18. Cell proliferation investigation of rBMSCs cultured on 30% SF/HA (dark in colour) and pure HA (light in colour) [185]

Figure 19. SEM images of attachment and proliferation of rBMSCs cultured on SF/HA scaffolds for 7, 14 and 21 days (a,c, e, g, i and k) scale bars = 100 µm; (b, d, f, h, j and l) scale bars = 50 µm [185]

Figure 20. Proliferation activities of cells cultured on pure SF, SF/nHA, and tissue culture dish (TCD) from 3 to 14 days. (p ≥ 0.05) indicates significant increase in proliferation activity [40]

Figure 21. ALP activity. Osteogenic differentiation of (a) rBMSCs on SF/HA scaffolds with 0 and 30 mol% HA, and (b) MC3TC-E1 on pure and mineralized SF/nHA after 5, 7, 10 and 14 days. ⋆ shows difference in ALP activity [40,185]

Figure 22. (a–d) In vivo biocompatibility of HA/SF-5% (black arrows denote lymphocytes, eosin and haematoxylin staining). (e–h) Increment of lymphocytes at the implantation site between control and experimental groups [187]

Figure 23. Photographs of rabbit bones on 12th week showing (a) no new bone formation without SF/HA-3, (b) new bone formation at bone defect site with SF/HA-3, and (c) new cortical bone formation and remodelling site with SF/HA-3 consisting BMSCs [188]

Figure 24. Radiographic illustration of gross anatomy of transplanted bone: structure of the defective radius at 12 weeks after operation in the experimental group (a) and the control group (b). Arrow indicates the transplantation site [189]

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