Smart scaffolds in bone tissue engineering: A systematic review of literature

Table 1 Description of included studies which modified the scaffolds by addition of growth factors

Ref.	Scaffold	Fabrication	Type of modification	Cell type	Tests and results
Yang et al[33], 2005	GTG + BMP-4	-	Surface modification	NRCOCs	ALP activity: ALP levels in GTG + BMP-4 samples higher than GTG samples H and E staining: Greater numbers of attached cells and richer matrix deposits in the GTG + BMP-4 samples VK staining: Larger mineralizing nodules, in greater numbers
Turhani et al[34], 2007	TCP + rhBMP-2	-	Surface coating	SaOS-2Cs	Cell viability and ALP activity: TCP + BMP-2 > TCP (control) OC secretion: TCP + BMP-2 = TCP
Abarrategi et al[29], 2008	β-TCP + rhBMP-2	Homogenized + demineralized + heterogeneous deacetylation	Surface coating	C2C12Cs	No alteration in biocompatibility In vivo: New bone formation 3 wk after surgery, much shorter time than control β-TCP ceramics
Fei et al[35], 2008	PLGA/CPC + rhBMP-2	Solvent-extraction technique	Surface coating	BMMSCs	ELISA: OC: PLGA/CPC + rhBMP-2: 1.2 ng/mL rhBMP-2/CPC: 0.4 ng/mL ALP activity: PLGA/CPC + rhBMP-2: 0.12 μg/h rhBMP-2/CPC: 0.06 μg/h
Yilgor et al[36], 2010	PCL + BMP-2/BMP-7	Plotting procedure Bioplotter’s CAD/CAM software + wet spinning	Scaffold architecture + surface coating	BMMSCs	ALP activity: BMP-2/BMP-7 + PCL: 1.2 nmol/min
Zhang et al[37], 2010	PCL + BMP-2	Crosslinking conjugation method	Surface coating	BMMSCs	RT-PCR: (relative) p-smad: PCL + BMP-2 conjugated five times higher than adsorption and control Col 1: PCL + BMP-2 conjugated: 3 PCL + BMP-2 adsorption: 1.5 Control (PCL): 1 ALP activity: 2 times higher than adsorption and control
Mitchell et al[38], 2010	Tol-OPN-ST+BMP2	-	Spatial and conformational display + surface coating	BMMSCs	Cell attachment: Tol-OPN-ST + BMP2: 175 mm3 Tol-OPN-ST: 60 mm3 Luciferase activity: Tol-OPN-ST + BMP2: 8000 ability unit Tol-BMP-ST: 6000 ability unit
Huh et al[39], 2011	Bio-Oss® + rhBMP-2 + iH	Deep and dry methods	Surface coating	MG63Cs	Cytotoxicity and proliferation: No difference compared to control (Bio-Oss®) ALP activity: 0.2 μmol/min per μg higher than control
Dai et al[40], 2011	CMMS + rhBMP-2	Polymeric sponge method	New composition + surface coating	BMMSCs	MTT assay: More viable cells on CMMS + rhBMP-2 compared to CMMS RT-PCR: (relative) RunX2: CMMS + rhBMP-2: 32 Control (CMMS): 4 OPN: CMMS + rhBMP-2: 38 Control: 3 In vivo: Induced the ectopic bone formation in the thigh muscle pouches of mice
Lu et al[41], 2012	Col/PLGA + CBD-BMP4	Forming collagen microsponges	Surface coating	BMMSCs	ALP activity: No differences compared to control group Scaffold supports cell adhesion and proliferation
Li et al[42], 2013	SWNTs-COOH/SWNTs-CH3 + BMP-2	Organic phase/aqueous phase replacement approach + sonication	Surface coating	C2C12Cs	ALP activity: (relative) SWNTs-ch3 + BMP-2: 150% SWNTs-cooh + BMP-2: 120%

TCP: Tricalcium phosphate; PCL: Poly(e-caprolactone); CMMS: Calcium/magnesium-doped mesoporous silica; CPC: Calcium phosphate cement; GTG: Glutaraldehydecrosslinked gelatin; PLGA: Poly(lactic-co-glycolic acid); col: Collagen; SWNTs-COOH and SWNTs-CH3: Hydrophilic COOH- and hydrophobic CH3-terminated single-walled carbon nanotubes; Tol-OPN-ST: TolAIII fusion-osteopontin-switch tag; rh: Recombinant human; BMP: Bone morphogenetic protein; CBD: Collagen-binding domain; BMMSCs: Bone marrow mesenchymal stem cells; NRCOCs: Neonatal rat calvaria osteoblast cells; MG63Cs: Human osteosarcoma cells; SaOS-2 Cs: Sarcoma osteogenic cells; C2C12C: Immortalized mouse myoblast cells; ALP: Alkaline phosphatase; H and E: Hematoxylin and eosin; VK: Von Kossa; RT-PCR: Real time polymerase chain reaction; ELISA: Enzyme-linked immunosorbent assay; MTT: 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide; XTT: Sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt; RunX2: Runt-related transcription factor 2; OPN: Osteopontin.

Table 2 Description of included studies which modified the scaffolds by addition of extracellular matrix molecules

Ref.	Scaffold	Fabrication	Type ofmodification	Cell type	Tests and results
Kim et al[43], 2006	BGNF + Col 1	Electrospinning process	Surface coating	MG63Cs	ALP activity: BGNF + Col 1 > Col 1
Lechner et al[44], 2006	β-TCP + F + T	-	New composition	BMMSCs	ALP activity: Increased during 28 d of culture FACS: CD 31: Increased expression by 100 folds
Turhani et al[34], 2007	HA + Col 1 + rhBMP-2	-	Surface coating	SaOS-2Cs	XTT proliferation assay: (relative) HA + col 1 + rhBMP-2: 1.5 Control: 0.5 ALP activity: HA + col 1 + rhBMP-2: 50 U/μg Control: 25 U/μg
Srouji et al[45], 2008	PCL + Col	Electrospun meshed scaffold	Electrospun nanofiber membrane	BMMSCs	Alamar Blue assay: PCL + Col = PCL [Data as mean ± SD (P < 0.05)] In vivo: Good integration after subcutaneous implantation
Hao et al[46], 2010	A: ADSCs-Col/PLGA-β-TCP B: Acellular Col/PLGA-β-TCP	Low- temperature deposition manufacturing (LDM) based on the layer-by-layer manufacturing principle of solid free-form fabrication	Surface coating	ADSCs	ALP activity: ECM mineralization in group A > group B Evident calcification level in group A, no apparent calcification in group B In vivo: Woven bone with a trabecular structure in group A No bone formation in group B
Xu et al[30], 2009	BG + Col-HYA-PS	Sol–gel method	New composite fabrication	BMMSCs	Cell attachment: Number of attached cells on BG + Col - HYA - PS was the highest Cell proliferation: BG + Col - HYA - PS > BG + Col and BG + Col - HYA > BG ALP activity: BG + Col - HYA - PS > BG + Col and BG + Col - HYA > BG
Kawai et al[47], 2009	OCP + Col	Mixing + lyophilization	Surface coating	MSST-2 Cs	Proliferation and attachment : OCP + Col > OCP (control) In vivo: OCP + Col: Enhanced bone regeneration ratio 83:17 generated maximum repair level of approximately 64% of the defect at 12 wk
Zhang et al[48], 2010	β-TCP + Col	Electrospun + impregnating methods	Architecture + surface coating	MG63Cs	MTT assay: (relative) β-TCP + Col: 0.30 Control (β-TCP): 0.25
Qu et al[49], 2010	C + HA + RGD peptide	In situ compositing hybridization + lyophilization	Surface coating	BMMSCs	Fluorescence microscopy: Cell adhesion rate: CS + HA: 54.7% C + HA + RGD peptide: 71.6% and 80.7% ALP activity: C + HA + RGD peptide: 0.00596 ± 0.00081 U/L per ng C + HA: 0.00283 ± 0.00025U/L per ng
Kang et al[50], 2011	Fi + HAH	The multi-head deposition system	Surface coating	ADSCs	ALP activity: Single day treatment: 0.5 mmol/mg No treatment: 0.5 mmol/mg Daily treatment: 3 mmol/mg
Marelli et al[51], 2011	nBG + DC	Plastic compression technique	Surface coating	MC3T3-E1 CS	Confocal microscopy of fluorescently: Attachment no difference ALP activity: nBG + DC: 3.5 × 105 Control (nBG): 2.5 × 105 Alamar blue assay: nBG + DC: 6.5 × 105 Control: 8 × 105
Phipps et al[52], 2011	PCL + Col I + nHA	Electrospun	Bone-mimetic electrospun matrices	BMMSCs	Activation of focal adhesion kinase: Cells seeded onto PCL/Col/nHA scaffolds were better spread, and exhibited greater amounts MTS assay: (relative) PCL + Col + nHA: 5 PCL + nHA: 2.5 PCL: 1
Weeks et al[53], 2012	PLLA + CXCL12, 13 + F + Col IV	-	Surface coating	BMMSCs	Antibody-blocking studies: Anti-α5β1 inhibits MSC attachment: PLLA + CXCL12, 13 + F + Col IV: 500 cells/mm2 PLLA + F + Col IV: 400 cells/mm2

BG: Bioglass; PLLA: Poly(l-lactic acid); Col: Collagen; C: Chitosan; PLGA: Poly(D,L-lactic-co-glycolic acid); PCL: Poly(e-caprolactone); HA: Hydroxy apatite; TCP: Tricalcium phosphate; HAH: Hyaluronic acid hydrogel; nBG: Nano-sized bioactive glass; BGNF: Bioactive glass nanofiber; OCP: Octacalcium phosphate; F: Fibronectin; Fi: Fibrin; nHA: Hydroxy apatite nano particles; CXCL: Chemokine ligand; T: Thrombin; rh: Recombinant human; BMP: Bone morphogenetic protein; HYA: Hyaluronic acid; PS: Phosphatidylserine; DC: Dense collagen; iH: Immobilized heparin; RGD: Arg-Gly-Asp; BMMSCs: Bone marrow mesenchymal stem cells; MG63Cs: Human osteosarcoma cells; SaOS-2 Cs: Sarcoma osteogenic cells; MC3T3-E1: Osteoblast precursor cells; ADSCs: Adipose-derived stem cells; MSST-2 Cs: Mouse bone marrow cells; ALP: Alkaline phosphatase; FACS: Fluorescent activated cell scan; XTT: Sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt; MTT: 2-(4,5-dimethyl-2 thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide; MTS: 5-[3-(carboxymethoxy)phenyl]-3-(4,5 dimethyl-2-thiazolyl)-2-(4-sulfophenyl)- 2H-tetrazolium inner salt; CD: Cluster of differentiation.

Table 3 Description of included studies which modified the scaffolds by addition of nanoparticles

Ref.	Scaffold	Fabrication	Type of modification	Cell type	Tests and results
Kim et al[54], 2006	PLGA + nHA	SC/PL and GF/PL	New composition fabrication	RCOCs	Average cell density: GF/PL = 2.4 × 106 cells/scaffold (86.5% increase) SC/PL = 2.1 × 106 cells/scaffold (69.7% increase) ALP activity: GF/PL= 0.6 mol/min per 106 SC/PL= 0.5 mol/min per 106
Wang et al[55], 2007	PA + nHA	Thermally induced phase inversion	Surface coating	BMMSCs	MTT assay and ALP activity: No negative effects on the BMMSCs in vitroIn vivo: Good biocompatibility and extensive osteoconductivity with host bone in vivo
Lv et al[56], 2009	PLGA + nHA	Microsphere sintering method (modification of the emulsion and solvent evaporation method)	New composition fabrication	BMMSCs	MTS assay: (cell number) PLGA + nHA : 1.2 million/mm2 PLAGA: 0.06 million/mm2 ALP activity: PLGA + nHA: 0.10 mL/μg PLAGA: 0 mL/μg
Roohani- Esfahani et al[57], 2010	BCP/PCL + nHA	Sonication	Surface coating	PHOLCs	ALP activity: BCP/PCL + nHA: 2 mmol/h per mg BCP: 0.5 mmol/h per mg RT-PCR: (relative) BSP: BCP/PCL + nHA: 0.4, PCL: 0 Runx2: BCP/PCL + nHA: 8, PCL: 5 OCN: BCP/PCL + nHA: 1.2, PCL: 0.8 Col I: BCP/PCL + nHA: 4.5, PCL: 2.5
Ye et al[58], 2010	PCL + nAP	Freeze-dried	Scaffold architecture	MG63Cs	Cell proliferation on composite scaffolds porosity of 76% > porosity of 53%
Phipps et al[52], 2011	PCL+ Col I + nHA	Electrospun	Bone-mimetic electrospun matrices	BMMSCs	Activation of focal adhesion kinase: Cells seeded onto PCL + col I + nHA scaffolds were better spread, and exhibited greater amounts MTS assay: (relative) PCL + Col + nHA: 5 PCL + nHA: 2.5 PCL: 1
Zhang et al[59], 2011	PLLA + ODA-nD	Sonication	Surface coating	7F2CS	Alamar Blue assay: A slight reduction in cell viability compared to control RT-PCR: (relative) ALP: PLLA + ND-ODA: 1, PLLA: 1 OCN: PLLA + ND-ODA: 3, PLLA: 2.8
Zeng et al[31], 2012	HA + MNPs	Tuning	New composition fabrication	MC3T3-E1Cs ROS 17/1.8Cs	MTT assay: HA + MNPs: MC3T3-E1: 0.2 OD value, ROS 17/1.8: 1.8 OD value HA: MC3T3-E1: 0.9, ROS 17/1.8: 0.25 ALP activity: HA + MNPs: MC3T3-E1: 0.75 U/mg, ROS 17/1.8: 4.5 U/mg HA: MC3T3-E1: 0.5 U/mg, ROS 17/1.8: 3.5 U/mg BGP activity: HA + MNPs: MC3T3-E1: 350 ng/L, ROS 17/1.8: 4000 ng/L HA: MC3T3-E1: 300 ng/L, ROS 17/1.8: 3500 ng/L
Hafezi et al[60], 2012	G + nBG	Homogenization through stirring	New composition fabrication	hAFCs	MTT assay: No difference compared to control In vivo: Radiographic evaluation: Improved the speed of the bone healing process
Buschmann et al[61], 2012	PLGA + n-aCaP	Electrospun	Electrospun PLGA/a-CaP scaffold architecture	ADSCs	MGTS: Extracellular matrix production was significantly higher FACS: CD13, CD29, CD44 and CD105 were expressed on PLGA + n-aCaP
Ganesh et al[62], 2012	nfPCL + nS	Electrospun	New composition fabrication	BMMSCs	FACS: CD29 = 3.3%, CD44 = 77.1%, CD73 = 94%, CD31, 34, 45 = 0% Cell viability: No difference compared to control BCA assay: NS + PCLN: 250 Ug/mg, PCLN: 100
Im et al[63], 2012	SWCNT + C + nHA	Lyophilization procedure	Scaffold architecture + new composition	BMMSCs	Cell adhesion and proliferation: SWCNT + C + nHA > SWCNT + C
Rodrigues et al[64], 2012	TCP + nfPCL	Electrospun + dynamic culturing environment	Scaffold architecture + new composition	BMMSCs	ELISA: TCP + nfPCL: 0.8 μg/mL TCP: 0.2 μg/mL ALP assay: TCP + nfPCL: 100 mol/h TCP: 20 mol/h
Panzavolta et al[65], 2013	G + nHA	Foaming + freeze-drying method	Surface coating+ scaffold architecture	BMMSCs	ALP activity: No difference compared to control RT-PCR: (Relative) ALP: G + nHA: 3.2, G: 1.8 Col I: G + nHA: 0.25, G: 0.25 Runx2: G + nHA: 0.5, G: 0.4 TGF-b1: G + nHA: 0.6, G: 0.55
Xing et al[66], 2013	PLLA + OTND	Manual perfusion technique under pressure	New composition fabrication	BMMSCs	BCA assay: No difference compared to control RT-PCR: (relative) OPN: PLLA + OTND: 3.5, PLL: 1 BSP: PLLA + OTND: 3, PLL: 1 BMP-2: PLLA + OTND: 4, PLL: 1 In vivo: New bone formation: PLLA + OTND: 50%, PLL: 10%
Liu et al[67], 2013	C + nHA	Electrospun	New composition fabrication	BMMSCs	Cell attachment: C + nHA: 1100 μm2 C: 250 RT-PCR: (Relative) BMP-2: C + nHA: 1.5, C: 1 BMP-4: C + nHA: 25, C: 0 Smad1: C + nHA: 9, C: 1 ALP: C + nHA: 1.2, C: 1 Runx2: C + nHA: 22, C: 1 Itga1: C + nHA: 17, C: 1 Itgb1: C + nHA: 7, C: 1 Itgb3: C + nHA: 8, C: 1 Myh9: C + nHA: 7, C: 1 Myh10: C + nHA: 3.5, C: 1 Col 1: C + nHA: 4.5, C: 1 In vivo: Superior ability of bone reconstruction
Wang et al[68], 2014	C + nHA	Lyophilization procedure + cold atmospheric plasma (CAP) treatment	Scaffold architecture + surface coating	BMMSCs	SEM: MSCs adhesion and infiltration were enhanced ELISA: (Relative) Fibronectin: C + nHA 0.8 compared to control (C) Vitronectin: C + nHA 1.1 compared to control

TCP: Tricalcium phosphate; PLLA: Poly(l-lactic acid); Col: Collagen; C: Chitosan; PCL: Poly(e-caprolactone); HA: Hydroxy apatite; G: Gelatin; PLGA: Poly(D,L-lactic-co-glycolic acid); GF/PL: Gas forming and particulate leaching; SC/PL: Solvent casting and particulate leaching; BCP: Biphasic calcium phosphate; SWNT: Single-walled carbon nanotubes; PA: Polyamide; nf: Nano fibrous; NHA: Hydroxy apatite nano particles; nAP: Nano apatite; OTND: Oxygen-terminated nanodiamond particles; nS: Nanoparticles of silica; n-aCaP: Amorphous calcium phosphate nanoparticles; MNPs: Magnetic nanoparticles; ODA-ND: Octadecylamine-functionalized nanodiamond; nBG: Nano-sized bioactive glass; BMMSCs: Bone marrow mesenchymal stem cells; MG63Cs: Human osteosarcoma cell line; MC3T3-E1: Osteoblast precursor cells; ADSCs: Adipose-derived stem cells; RCOCs: Rat calvarial osteoblasts cells; hAFCs: Human airway fibroblast cells; ROS 17/1.8 Cs: Osteoblastic cells; 7F2CS: Mouse bone marrow cells; PHOLCs: Primary human osteoblasts-like cells; ALP: Alkaline phosphatase; MTT: 2-(4,5-dimethyl-2 thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide; MTS: 5-[3-(carboxymethoxy)phenyl]-3-(4,5 dimethyl-2-thiazolyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt; FACS: Fluorescent activated cell scan; RT-PCR: Reverse transcriptase polymerase chain reaction; ELISA: Enzyme-linked immunosorbent assay; SEM: Scanning electron microscope; BGP: Bone Gla protein; BCA: Bicinchoninic acid; MGTS: Masson Goldner Trichrome staining; CD: Cluster of differentiation; Itga1: Integrin a1; Itgb1: Integrin b1; Itgb3: Integrin b3; Myh9: Non-muscle myosin 9; Myh10: Myosin 10.