Investigational growth factors utilized in animal models of spinal fusion: Systematic review

Table 1 Search terms across 4 databases to identify experimental growth factors in animal models of spinal fusion

Databases	Search terms
PubMed (1693)	(spinal fusion [mesh] OR spine fusion*[tw] OR spinal fusion*[tw] OR “spondylosyndesis”[tw]) AND (animals [mesh:noexp] OR “chordata”[mesh:noexp] OR (“vertebrates”[mesh] NOT “humans”[mesh]) OR “animals, domestic”[mesh] OR “animals, exotic”[mesh] OR “animals, genetically modified”[mesh] OR “animals, laboratory”[mesh] OR “animals, outbred strains”[mesh] OR “animals, wild”[mesh] OR “animals, zoo”[mesh] OR “mice”[tw] OR “mouse” [tw] OR murine*[tw] OR “rat”[tw] OR “rats”[tw] OR rabbit*[tw] OR “leporine”[tw] OR ovine*[tw] OR sheep*[tw] OR goat*[tw] OR “caprine”[tw] OR “porcine”[tw] OR “pig” [tw] OR “pigs”[tw] OR “swine”[tw] OR “cow”[tw] OR “cows”[tw] OR “bovine”[tw] OR “horse”[tw] OR “horses”[tw] OR equine*[tw] OR “canine”[tw] OR “feline”[tw] OR “animal”[tw] OR “animals” [tw] OR “dog”[tw] OR “dogs”[tw] OR “cat”[tw] OR “cats”[tw] OR monkey*[tw] OR “non human primate”[tw] OR “non human primates”[tw] OR “simian”[tw] OR “ape”[tw] OR “apes”[tw] OR “gorilla”[tw] OR “gorillas”[tw] OR “piscine”[tw] OR “fish”[tw] OR “fishes”[tw] OR “goose”[tw] OR “geese”[tw] OR “fowl”[tw] OR “poultry”[tw] OR “chicken”[tw] OR “chickens”[tw])
Embase (1709)
Cochrane Library (52)
Web of Science (1352)
Total Results (4806)

Table 2 Normal biological activity of each included growth factor

Growth factors	Normal biologic activity[16-31]
AB204	Chimera of activin A and BMP-2 - which are both members of the transforming growth factor-beta (TGF-beta) superfamily
Ang-1	Pro-angiogenic growth factor that mediates reciprocal interactions between the endothelium and surrounding matrix, inhibits endothelial permeability, and contributes to blood vessel maturation and stability
Calcitonin	Secreted by the parafollicular (C cells) of the thyroid gland and is a direct inhibitor of osteoclasts
EPO	Produced in the kidney in response to hypoxia and is a well-known growth factor essential for hematopoiesis
bFGF	Broad mitogenic and angiogenic functions and is important for limb and nervous system development, wound healing, and tumor growth
GDF-5	Structurally similar to BMP-2 and BMP-7, GDF-5 is a secreted member of the TGF-beta superfamily of proteins involved in the development of various tissues and cell types, as well as the growth of neuronal axons and dendrites
IGF-1/TGF-beta	IGF-1 is a protein with similar structure and function to insulin involved in mediating growth and development. TGF-beta 3 is a secreted ligand capable of binding to various TGF-beta receptors involved in embryogenesis and cell differentiation and may play a role in wound healing
Insulin	A product of post-translational modification of proinsulin, involved in intracellular glucose uptake
NELL-1	Secreted protein containing epidermal growth factor-like repeats. It binds to the cell surface heterodimer integrin α3β1, resulting in intracellular changes that induce osteoblastogenic programming. Its overexpression is associated with craniosynostosis
Noggin	Secreted polypeptide which binds and inactivates members of the transforming growth factor-beta superfamily of proteins. Noggin is important for developmental process such neural tube closure and joint formation, and when mutated can lead to proximal symphalangism and multiple synostoses syndrome
P-15	Synthetic 15-amino acid peptide with an identical sequence to the cell-binding domain found on the α1(I) chain of Type-I collagen. It is combined with an anorganic bovine-derived hydroxyapatite matrix (ABM) to produce an osteoinductive and osteoconductive bone graft alternative
Peptide B2A	Synthetic, receptor-targeted peptide that cooperatively enhances biologic BMP-2 response
SPP24	Secreted bone matrix protein that belongs to the cystatin superfamily and binds proteins in the transforming growth factor-beta family of cytokines

BMP: Bone morphogenetic protein; GDF: Growth differentiation factor; TGF: Transforming growth factor; IGF: Insulin-like growth factor.

Table 3 Demographic characteristics for all included studies

Animal model	Studies (n)
Dog	1[32]
Rat	8[33,36,38,47-49,51,56]
Rabbit	7[34,35,37,40,42,52,54]
Macaque	1[20]
Sheep	7[41,43-45,50,53,55]
Baboon	1[39]
Goat	1[46]
Spinal levels fused
C3 – C4	4[43-46]
L1 – L2	1[32]
L2 – L3	1[55]
L3 – L4	3[20,50,56]
L4 – L5	12[32,35,36,38,39,41,47-49,51,54,55]
L3 – L5	2[33,53]
L5 – L6	7[20,34,37,40,42,50,52]
L6 – L7	1[35]
Spinal region
Cervical	4[43-46]
Lumbar	22[20,32-42,47-56]
Biomechanical location
Mobile segments	11[20,32,33,43-46,50,53,55,56]
Junctional segments	21[20,32-42,47-55]
Number of levels fused
1-Level fusion	19[34,36-49,51,52,54,56]
Two separate 1-level fusions	5[20,32,35,50,55]
2-Level fusion	2[33,53]
Surgical approach
Anterior	8[20,43-46,50,53,55]
Posterior	18[32-42,47-49,51,52,54,56]

Table 4 Experimental models and rates of bony fusion for each included study

Growth factor	Animal(n)	Levels (a/p)1	Experimental groups	Fusion rate
AB204
Zheng et al[32], 2017	Dog (n = 56)	L1-L2, L4-L5 (p)	(1) BCP; (2) BCP + rhBMP-2; (3) BCP + AB204	(1) 6.3%; (2) 15%; (3) 90%2
COMP-Ang-1
Park et al[33], 2011	Rat (n = 56)	L3-L5 (p)	(1) ICBG; (2) ICBG + bovine serum albumin-impregnated collagen sponge; (3) ICBG + COMP-Ang-1-impregnated collagen sponge	(2) 38.9%; (2) 42.1%; (3) 89.5%2
Calcitonin
Babat et al[34], 2005	Rabbit (n = 56)	L5-L6 (p)	(1) ICBG; (2) ICBG + calcitonin; (3) ICBG + pamidronate	(1) 56%; (2) 68%; (3) 37%
Liu et al[35], 2012	Rabbit (n = 32)	L4-L5, L6-L7 (p)	(1) ICBG; (2) ICBG + calcitonin; (3) ICBG + interspinous fixation (steel wire); (4) ICBG + interspinous fixation (steel wire) + calcitonin	(1) 75%; (2) 100%; (3) 75% 100%
Liu et al[36], 2015	Rat (n = 50)	L4-L5 (p)	(1) Sham surgery + saline vehicle; (2) Ovariectomy + saline vehicle; (3) Spinal fusion (ICBG) + saline vehicle; (4) Ovariectomy + Spinal fusion (ICBG); (5) Ovariectomy + Spinal fusion (ICBG) + calcitonin	Not reported
EPO
Rolfing et al[37], 2011	Rabbit (n = 28)	L5-L6 (p)	(1) ICBG + Epoetin beta subcutaneous injection; (2) ICBG + Saline subcutaneous injection	(1) 86%; (2) 71%
bFGF
Inoue et al[38], 2017	Rat (n = 20)	L4-L5 (p)	(1) Allograft; (2) Allograft + bFGF	Not reported
GDF-5
Spiro et al[39], 2000	Baboon (n = 36)	L4-L5 (p)	(1) ICBG; (2) Collagen matrix strips; (3) Collagen matrix strips + rhGDF-5 (500μg/cm3); (4) Collagen matrix strips + rhGDF-5 (1500μg/cm3)	(1) 22%; (2) 0%; (3) 44%; (4) 11%
Spiro et al[40], 2001	Rabbit (n = 31)	L5-L6 (p)	(1) ICBG; (2) Hydroxyapatite-mineralized collagen matrix (Matrix); (3) Matrix + bone marrow; (4) Healos strips + rhGDF-5 (0.1 mg/cc); (5) Healos strips + rhGDF-5 (1.0 mg/cc); (6) Non-crosslinked collagen strips + rhGDF-5 (0.1 mg/cc); (7) Non-crosslinked collagen strips + rhGDF-5 (1.0 mg/cc); (8) Collagen fiber slurry + rhGDF-5 (0.1 mg/cc); (9) Collagen fiber slurry + rhGDF-5 (1.0 mg/cc)	(1) 33%; (2) 0%; (3) 0%; (4) 0%; (5) 67%; (6) 75%; (7) 80%; (8) 25%; (9) 0%
Jahng et al[41], 2004	Sheep (n = 8)	L4-L5 (p)	(1) ICBG; (2) Healos + GDF-5	(1)100%; (2) 100%
Magit et al[42], 2006	Rabbit (n = 65)	L5-L6 (p)	(1) ICBG; (2) Healos; (3) Healos + rhGDF-5 (0.5 mg/cc); (4) Healos + rhGDF-5 (1 mg/cc); (5) Healos + rhGDF-5 (1.5 mg/cc)	(1) 38%; (2) 0%; (3) 100%2; (4) 100%2; (5) 100%2
IGF-1/TGF-β
Kandziora et al[43], 2002	Sheep (n = 32)	C3-C4 (a)	(1) Titanium cage (Cage); (2) Cage + ICBG; (3) Cage + PDLLA + BMP-2; (4) Cage + PDLLA + rh-IGF-1/TGF-β	(1) 0%; (2) 13%; (3) 13%; (4) 13%
Kandziora et al[44], 2002	Sheep (n = 32)	C3-C4 (a)	(1) ICBG; (2) Titanium cage (Cage); (3) Cage + PDLLA; (4) Cage + PDLLA + rh-IGF-1/TGF-β	(1) 0%; (2) 0%; (3) 0%; (4) 12.5%
Kandziora et al[45], 2003	Sheep (n = 32)	C3-C4 (a)	(1) Titanium cage + PDLLA (Cage); (2) Cage + rh-IGF-1/TGF-β (2.5/0.5%); (3) Cage + rh-IGF-1/TGF-β (5/1%); (4) Cage + rh-IGF-1/TGF-β (10/2%)	Not reported
Gu et al[46], 2006	Goat (n = 32)	C3-C4 (a)	(1) ICBG; (2) Titanium cage (Cage); (3) Cage + hydroxyapatite; (4) Cage + IGF-1/TGF-β (5/1%)	(1) 0%; (2) 25%; (3) 38%; (4) 63%
Insulin
Koerner et al[47], 2013	Rat (n = 19)	L4-L5 (p)	(1) ICBG + 100% palmitic acid; (2) ICBG + Linplant (95% palmitic acid and 5% bovine insulin)	(1) 11%; (2) 60%2
NELL-1
Lee et al[48], 2009	Rat (n = 10)	L4-L5 (p)	(1) Demineralized bone putty (Putty) + PBS; (2) Putty + rh-NELL-1	(1) 0%; (2) 60%
Li et al[49], 2010	Rat (n = 24)	L4-L5 (p)	(1) DBX + PBS; (2) DBX + NELL-1 (2.5 g); (3) DBX + NELL-1 (5 μg)	(1) 25%; (2) 75%; (3) 88%
Siu et al[50], 2011	Sheep (n = 32)	L3-L4 L5-L6 (a)	(1) Cage + DBM; (2) Cage + inactivated DBM; (3) Cage + DBM + NELL-1 (0.3 mg/mL) Cage + DBM + NELL-1 (0.6 mg/mL); (4) Cage + inactivated; (5) DBM + NELL-1 (0.3 mg/mL); (6) Cage + inactivated DBM + NELL-1 (0.3 mg/mL)	(1) 50%; (2) 50%; (3) 87.5%2; (4) 100%2; (5) 100%2; (6) 100%2
Yuan et al[51], 2013	Rat (n = 26)	L4-L5 (p)	(1) DBX + PBS; (2) DBX + NELL-1 (10μg); (3) DBX + NELL-1 (50μg); (4) Acellular collagen sponge (ACS) + PBS; (5) ACS + BMP-2; (6) ICBG	(1) 20%; (2) 100%; (3) 100%; (4) 0%; (5) 100%; (6) 0%
James et al[20], 2017	Macaque (n = 12)	L3-L4 L5-L6 (a)	(1) Cage + aTCP; (2) Cage + DBX + rh-NELL-1-loaded aTCP (1mg/mL); (3) Cage + DBX + rh-NELL-1-loaded aTCP (1.7mg/mL)	(1) 25%; (2) 25%; (3) 100%
Noggin
Klineberg et al[52], 2014	Rabbit (n = 25)	L5-L6 (p)	(1) ICBG + Noggin scrambled siRNA bilateral; (2) ICBG + scrambled siRNA one side + siRNA other side; (3) ICBG + functional siRNA bilateral	(1) - (2) - (3) 50%
P-15
Sherman et al[53], 2010	Sheep (n = 12)	L3-L5 (a)	(1) PEEK + ICBG; (2) PEEK + anorganic bovine-derived matrix/P-15	(1) 83%; (2)100%
Peptide B2A
Smucker et al[54], 2008	Rabbit (n = 45)	L4-L5 (p)	(1) ICBG; (2) ICBG + TCP (1:1); (3) ICBG + B2A (50 μg); (4) ICBG + B2A (100 μg); (5) ICBG + B2A (300 μg)	(1) 25%; (2) 22%; (3) 56%; (4) 78%2 (vs A and B); (5) 40%
Cunningham et al[55], 2009	Sheep (n = 40)	L2-L3 L4-L5 (a)	(1) PEEK + ICBG; (2) PEEK + ICBG + B2A (50 μg); (3) PEEK + ICBG + B2A (100 μg); (4) PEEK + ICBG + B2A (300 μg); (5) PEEK + ICBG + B2A (600 μg)	(1) 63%; (2) 88%; (3) 88%; (4) 88%; (5) 75%
SPP24
Sintuu et al[56], 2011	Rat		(1) Collagen sponge (Sponge); (2) Sponge + rhBMP-2; (3) Sponge + rhBMP-2 (1 μg) + SPP24 (100 μg); (4) Sponge + rhBMP-2 (1 μg) + SPP24 (500 μg); (5) Sponge + rhBMP-2 (1 μg) + SPP24 (1 mg); (6) Sponge + rhBMP-2 (1 μg) + SPP24 (2.5 mg); (7) Sponge + rhBMP-2 (1 μg) + SPP18 (100 μg); (8) Sponge + rhBMP-2 (1 μg) + SPP18 (500 μg); (9) Sponge + rhBMP-2 (1 μg) + SPP18 (1 mg); (10) Sponge + rhBMP-2 (1 μg) + SPP18 (2.5 mg); (11) Sponge + rhBMP-2 (10 μg) + SPP24 (100 μg); (12) Sponge + rhBMP-2 (10 μg) + SPP24 (500 μg); (13) Sponge + rhBMP-2 (10 μg) + SPP24 (1 mg); (14) Sponge + rhBMP-2 (10 μg) + SPP24 (2.5 mg); (15) Sponge + rhBMP-2 (10 μg) + SPP18 (100 μg); (16) Sponge + rhBMP-2 (10 μg) + SPP18 (500 μg); (17) Sponge + rhBMP-2 (10 μg) + SPP18 (1 mg); (18) Sponge + rhBMP-2 (10 μg) + SPP18 (2.5 mg)	(1) 0%; (2) 100%2; (3) 0%; (4) 0%; (5) 0%; (6) 0%; (7) 0%; (8) 0%; (9) 0%; (10) 0%; (11) 0%; (12) 0%; (13) 0%; (14) 0%; (15) 0%; (16) 0%; (17) 0%; (18) 0%

¹(a/p): anterior/posterolateral surgical approach; N²: Statistically significant compared to all other experimental groups. ICBG: Iliac crest bone graft. BCP: Biphasic calcium phosphate; rhBMP-2: Recombinant human bone morphogenetic protein 2; bFGF: Basic fibroblast growth factor; GDF-5: Growth differentiation factor 5; PDLLA: Poly-D, L-lactide; TGF: Transforming growth factor; IGF: Insulin-like growth factor.