Table 1: Studies on CS-based scaffolds for bone tissue regeneration.

AuthorsScaffold typeStudy outlineResultsConclusion

Miranda et al. 2011 [8]CS (DA: 15%; MW: NA) +GEL (3 : 1 ratio) as membranes (2D) or sponges (3D) crosslinked with glutaraldehydeIn vitro: CC in BMMSCs culture
In vivo: 8-week-old Lewis rats had the dental alveoli filled with scaffolds and analysed by HT
Sample (in vivo):
Cell proliferation and osteogenic differentiation In vitro  
In vivo: acute inflammation 
Bone regeneration
Slow biodegradation
CS+GEL sponges demonstrated biocompatibility and potential for application in bone tissue engineering

Danilchenko et al. 2011 [41]CS (low MW and DA 15–25%) + HA composite sponges (at 15 : 85, 30 : 70, 50 : 50, and 80 : 20 ratios)In vivo: implantation in tibial defects of 4-month-old rats; HT evaluation, 
SEM and microhardness of tibias; serum bone-specific alkaline phosphatase (BAP)
Control group: tibial defects without scaffolds
Sample:
Complete biodegradation after 24 days, promotion of bone regeneration
However the amount and speed of newly formed bone tissue were no different statistically from the controls
The scaffolds demonstrated biocompatibility and osteoconductive potential

Niu et al. 2011 [42]CS microspheres (DA 15–25%; MW: NA
viscosity: 200 cps) encapsulated with BMP-2 and incorporated with nHA-COL-PLLA (ratio: NA)
In vitro: CC of MC3T3-E1 mouse osteoblastic cell culture; 
In vivo: implantation of scaffolds in femoral defects of New Zealand white rabbits; HT and radiographic analyses
Control group: nHA-COL-PLLA 
Sample: not specified
Increased cell activity of osteoblasts on the CS+BMP-2 scaffold Increased radiographic density in the group with CS+BMP-2 and a far better repair than with the controls after 4 weeksCS microspheres demonstrated great potential for use as BMP-2 matrix carrier for bone regeneration (in vitro)

Costa-Pinto et al. 2012 [43]CS (DA: NA; MW: NA) combined with PBS (1 : 1 ratio), with or without SCIn vitro: human BMMSCs culture and evaluation of osteogenic differentiation 
In vivo: implantation of scaffolds in calvarial defects of 7-week-old mice; micro-CT of the bone regeneration 
Control group: untreated defects 
Sample:
In vitro: osteogenic proliferation and differentiation on CS scaffolds  
In vivo: the scaffolds were effective in regenerating calvarium bone tissue (better results in group with SC)
The CS-based scaffolds were shown to be biocompatible and promoted bone regeneration in vivo, particularly in the presence of SC

Hou et al. 2012 [44](i) COL sponges
(ii) COL sponges with BMP2
(iii) COL sponges with CS microspheres (MW of 90 kDa; DA 5%) and BMP2 (ratio: NA)
In vitro: BMP-2 release tests
In vivo: implantation of sponges in radius defects of New Zealand white rabbits; micro-CT and HT evaluations; 3-point bending test of the regenerated bones for mechanical evaluation  
Control group: normal bone 
Sample:
In vitro: COL+CS+BMP2 produced a slower, more gradual release up to 35 days 
In vivo: COL+CS+BMP-2 demonstrated better bone regeneration, complete closure of defects (12 weeks) and greater mechanical performance of newly formed bone tissue
Addition of CS improved release of BMP2, promoted better bone regeneration, and increased mechanical performance of regenerated bone ()

Zhang et al. 2012 [45]CS in gel (DA: NA; MW: NA), either pure or in a composite with nHA (ratio: NA)In vivo: implantation in defects of the femoral condyle of New Zealand white rabbits; CT, macroscopic, and HT analyses of the defects   
Control group: untreated defects 
Sample: ; (control)
The CS+nHA group demonstrated greater bone neoformation than the CS and control groups, and complete repair of the defects after 12 weeks. Pure CS was better than the controlCS+nHA has potential for use in bone regeneration of critical defects, favoring new bone formation when compared to CS alone ()

Miranda et al. 2012 [46]CS (DA 15%; MW; NA) + GEL crosslinked with glutaraldehyde and incorporated with BMMSC (ratio: NA)In vivo: implantation in fresh tooth sockets of Lewis rat molars; CT, HT, and IHC analyses 
Control group: contralateral untreated tooth sockets 
Sample:
CS+GEL+SC group presented greater bone formation after 21 and 35 days, with newly formed bone tissue with a greater level of maturity. There was no control with pure CSThere was greater alveolar bone maturation after extraction with the use of CS+GEL+SC ()

Florczyk et al. 2013 [47]CS (DA: NA; MW: NA) +ALG sponges incorporated with BMMSC or BMP-2  
(ratio: NA)
In vitro: CC in BMMSC culture of rats  
In vivo: implantation of scaffolds in critical calvarial defects of Sprague-Dawley rats; micro-CT, HT, and IHC analyses  
Control group: untreated defects  
Sample:
CS+ALG+BMP-2 demonstrated the highest percentage of defect closure, expression of markers, and bone regeneration of all the groups, after 16 weeks All groups showed better results than the controlCS+ALG were biocompatible and permitted osteogenic growth and SC differentiation In vitro and presented osteoconductive properties in vivo ()

Jiang et al. 2013 [48]CS+CMC (1 : 1 ratio) membranes and nHA (0, 20, 40 or 60 wt%).
DA: NA; MW: NA
In vitro: CC and osteogenic differentiation in osteoblast cell culture; evaluation of biodegradation  
In vivo: implantation in long defects in the radius of rabbits; radiographic, micro-CT, and HT analyses  
Control group: untreated defects 
Sample
In vitro: CS+CMC showed faster degradation; pure CS degraded more slowly; greater cell proliferation and osteogenic differentiation with CS+CMC+nHA (60% wt)  
In vivo: there was bone regeneration of defects after 12 weeks even in the control, but with greater bone volume in the CS+CMC+nHA group
Cylindrical/spiral CS+CMC+nHA scaffold demonstrated biomimetic behavior, promoting cell adhesion, proliferation, and differentiation In vitro and bone regeneration in vivo

Jia et al. 2014 [28](i) CS sponges (MW 100–300 kDa; DA 6.63%) 
(ii) CS sponges incorporated with osteogenesis and/or angiogenesis inducing genetic factors (RNA)
In vitro: RNA release tests; osteogenic proliferation and differentiation of rat BMSC  
In vivo: Implantation of scaffolds in calvarial defects of rats  
Micro-CT analyses  
Control groups: pure CS and CS with RNA negative control 
Sample: not specified
In vitro: CS+ both RNAs exhibited greater cell proliferation and osteogenic differentiation than controls  
In vivo: CS+ both RNAs promoted increase in area of newly formed bone after 3 months compared to controls
CS sponges impregnated with two RNA factors promoted greater in vitro osteogenesis and angiogenesis and bone regeneration in defects of rat calvarias than pure CS ()

Cao et al. 2014 [49](i) GEL sponge (Gelfoam®)
(ii) Gelfoam with BMP-2  
(iii) Gelfoam with sulfonated CS (MW 98 kDa; DA: NA) + BMP-2 (ratio: 1 : 1)  
(iv) Gelfoam with sulfonated CS + BMP2 in nanoparticles
In vitro: CC, osteogenic, and angiogenic differentiation in culture of human umbilical vein endothelial cells  
In vivo: implantation in long defects in the radius of 5-mo. New Zealand rabbits; micro-CT, HT, and micro-angiography analyses; 3-point bending test for mechanical testing 
Control groups: (Gelfoam) and normal bone
In vitro: greater cell proliferation and viability in the groups with CS+BMP (nanoparticles)   
In vivo: better regeneration and angiogenesis in animals with CS+BMP (nanoparticles); mechanical performance was similar to normal bone
CS+BMP in nanoparticles incorporated in GEL promoted greater neovascularization and bone regeneration In vitro and in vivo than GEL alone or with BMP (), showing potential for bone and vascular regeneration

Lee et al. 2014 [50]CS (MW310 kDa; DA 10%) + HA or nano-HA composites (ratio: NA)In vitro: CC in cell culture of MC3T3-E1 preosteoblasts 
In vivo: grafts in segmentary tibial defects of New Zealand rabbits; evaluation via micro-CT and HT  
Control groups: NA 
Sample: to 8
In vitro: CS+nHA demonstrated greater cell proliferation and viability 
In vivo: better histological and radiographic results with discrete ossification in the nHA+CS group
CS+nHA demonstrated potential for application in bone regeneration

Fernandez et al. 2014 [51]Composite as a paste of CS (DA: NA; MW: NA) and a bioceramic of βTCP+CaO+ZnO  
(ratio: 60 : 40)
In vivo: implantation of scaffolds in critical calvarial defects of 4-mo. Wistar rats; HT and histomorphometric analyses  
Control groups: untreated defects 
Sample:
Scaffolds showed bone regeneration after 40 days, with formation of bone marrow, vessels, and avascular cortical bone and complete closure of the defects by day 60 
Control results not specified
CS+βTCP+CaO+ZnO promoted osteoinduction and neovascularization of the bone defects, showing potential for bone regeneration

Fan et al. 2014 [52]Composite sponges of CS (MW 255 kDa; DA 15–25%) + Condroitin sulfphate (ratio: 2 : 1) coated with HA;  
The sponges were used with or without SC and/or BMP-2
In vitro: CC in adipose-derived SC; BMP-2 release assay  
In vivo: implantation in critical defects in the jaws of rats; analyses via micro-CT, immunofluorescence, and HT  
Control groups: NA 
Sample:
In vivo: greater bone formation in the CS+HA+BMP-2+SC group, with greater expression of collagen and osteocalcin, compared to blank scaffoldsCS+BMP2+CS demonstrated great potential for the regeneration of bone defects, with a synergistic effect of the combination ()

Koç et al. 2014 [53]CS sponges (MW 400 kDa; DA < 15%) + HA (ratio: 9 : 1), whether or not activated with VEGFIn vitro: CC and VEGF secretion in osteoblast culture  
In vivo: implantation in epigastric fasciovascular flaps of Wistar rats; HT and IHC analyses 
Control groups: untreated flaps and blank scaffolds 
Sample:
In vitro: CS+HA+VEGF: greater proliferation of osteoblasts and secretion of VEGF 
In vivo: CS+HA+VEGF +osteoblasts showed greater neovascularization and ectopic bone formation, at 28 days compared to blank scaffolds  
Control results not specified
CS+HA+VEGF promoted proliferation of human osteoblasts, induction of ectopic bone formation, and vascular neoformation ()

Lai et al. 2015 [54]Nanofibrous membranes of CS (MW: 100 kDa; DA 2%) and SF (ratio 1 : 1) + nHA (10% or 30%), either with or without stem cells.In vitro: CC and osteogenic differentiation of BMMSC on CS/SF with or without nHA  
In vivo: subcutaneous implantation of CS/SF/nHA30%/BMMSC in mice; HT and IHC analyses  
Control group: acellular scaffold  
Sample: not specified
In vitro: CS+SF+nHA30% exhibited greater osteogenic differentiation  
In vivo: only CS+SF+nHA with SC induced formation of ectopic osteoid tissue after 8 weeks
The CS+SF+nHA scaffold favored osteogenic proliferation and differentiation in vitro () and when combined with SC, induced bone formation in vivo

Ghosh et al. 2015 [55]CS (MW 710 kDa; DA < 10%) crosslinked or otherwise, with citric acid and/or carbo-di-imidesIn vitro: CC and osteogenic differentiation in culture of BMSC  
In vivo: implantation in tibial defects of rabbits; HT analysis of bone regeneration
Control groups not specified  
Sample: not specified
In vitro: crosslinked CS with citric acid demonstrated greater osteogenic adhesion, proliferation and differentiation  
In vivo: dual crosslinked CS exhibited greater deposition of collagen and bone regeneration after 6 weeks
The dual crosslinked CS scaffold demonstrated greater cytocompatibility in vitro and bone regeneration in vivo ()

Caridade et al. 2015 [56]CS membranes (MW 770 kDa; DA 22%) +ALG, crosslinked with carbo-di-imides and incorporated or otherwise with BMP-2 (ratio: NA)In vitro: CC and myogenic and osteogenic differentiation 
In vivo: implantation in subcutaneous tissue of mice and evaluation via micro-CT
Control groups: not specified
Sample:
In vitro: CS+BMP-2 induced osteogenic differentiation and release of BMP-2 
In vivo: only the most crosslinked membranes were capable of inducing osteogenesis at 52 days
Crosslinked CS+BMP-2 have potential for use as a periosteum substitute for bone regeneration

Frohbergh et al. 2015 [57]Microfibers of genipin crosslinked CS (DA 15–25%; medium MW), with or without nHA and SC (ratio: NA)In vitro: CC and osteogenic differentiation in murine MSC culture  
In vivo: implantation in calvarial defects of 4–6-w mice; HT and micro-CT analyses  
Control group: untreated defects 
Sample:
In vitro: CS+nHA produced twice the osteogenic differentiation of CS  
In vivo: CS+nHA+SC exhibited greater bone neoformation than any of the others, after 3 months
CS crosslinked with genipin has potential for use in bone regeneration; addition of nHA and stem cells increased bone regeneration in vivo ()

Dhivya et al. 2015 [58]Hydrogels of CS-Zn (DA: NA; MW: NA)+β-glycerophosphate + nHA (ratio: 8 : 1; 1) or without nHAIn vitro: cell proliferation and differentiation in mouse MSC culture 
In vivo: insertion into tibial defects of Wistar rats; radiographic and HT analyses  
Control group: untreated defects  
Sample: not specified
In vitro: scaffolds favored osteoblast proliferation and differentiation  
In vivo: greater mineralization and formation of collagen after 14 days in scaffolds with nHA
CS-Zn + β-glycerophosphate demonstrated bone regeneration potential; addition of hydroxyapatite promoted and accelerated bone formation ()

D’Mello et al. 2015 [59]Sponges of CS (MW: 110 to 150 kDa; DA: NA), whether or not incorporated with copper sulfate (ratio: NA)In vivo: implantation in calvarial defects of 14-week-old Fisher rats; analyses via micro-CT and HT  
Control groups: untreated defects 
Sample: (control )
CS + copper exhibited greater bone neoformation than pure CS or control, both via micro-CT and via histological analysesCS + copper has great potential for application in bone regeneration and promoted bone regeneration in vivo ()

Ji et al. 2015 [60]3D disks of CS (low MW; DA: NA) +GEL with spherical or cylindrical nHA 
(ratio: 1 : 1 : 3) with or without SC.
In vitro: morphology, osteogenic proliferation, and differentiation of human gingival fibroblast-derived induced pluripotent SC 
In vivo: implantation of scaffolds with or without SC in subcutaneous tissue of mice; HT and IHC analyses of ectopic bone-like tissue formation  
Control group: not specified 
Sample:
In vitro: scaffolds with spherical nHA demonstrated greater osteogenic proliferation and differentiation ()  
In vivo: CS+GEL+ nHA+SC showed greater bone-like tissue formation than acellular scaffolds (); spherical nHA induced thicker bone-like formation after 12 weeks ()
CS+GEL+spherical nHA combined with pluripotent human cells induced ectopic bone-like tissue formation and represent an innovative approach with the potential for application in bone tissue engineering

Shalumon et al. 2015 [61]Nanofibrous membranes of CS (MW 100 kDa; DA 2%) +SF+nHA+BMP2, whether or not impregnated with SC 
(ratio: NA)
In vitro: osteogenic proliferation and differentiation of MSC; BMP-2 release test
In vivo: implantation of scaffolds with or without MSC in subcutaneous tissue of 6–8-week-old mice; HT and IHC analyses after 4 and 8 weeks  
Control groups: not specified 
Sample:
In vitro: BMP-2 increased osteogenic differentiation of MSC on CS+SF and CS+SF+nHA scaffolds  
In vivo: cellular or acellular scaffolds were capable of inducing formation of ectopic bone-like tissue, with greater intensity when SC was present
CS+SF+nHA scaffolds with BMP-2 induced greater osteogenic differentiation In vitro () and showed great potential for application in bone regeneration in vivo

Xie et al. 2016 [62]Nanofibers of CS (DA < 15%; MW: NA)+HA (ratio: 7 : 3) and/or COL+SCIn vitro: CC and osteogenic differentiation of induced pluripotent SC+ MSC  
In vivo: implantation scaffolds with or without SC in critical calvarial defects of 6-week-old mice; HT analysis (4, 6, and 8 weeks) and tomographic analysis (6 weeks)  
Control group: untreated defects, pure CS, and TCP 
Sample: (histology); (CT)
In vitro: CS+HA+COL promoted greater osteogenic differentiation than CS, CS+HA, and TCP 
In vivo: CS+HA+COL with SC promoted greater bone neoformation, via CT and histology, with complete regeneration of defects
CS+COL+HA with stem cells promoted effective bone neoformation in vitro and in vivo, with better results than controls (), showing potential for bone regeneration in clinical applications

ALG: alginate; BAP: bone alkaline phosphatase; BMMSCs: bone marrow mesenchymal stem cells; BMP2: type 2 morphogenetic bone protein; CC: cytocompatibility; CMC: carboxymethyl cellulose; COL: collagen; CS: chitosan; CT/micro-CT: computed tomography/micro-computed tomography; DA: degree of acetylation; GEL: gelatin; HA/nHA: hydroxyapatite/nanohydroxyapatite; HT: histological; kDa: kilodaltons; MSC: mesenchymal stem cells; MW: molecular weight; NA: not available; PLLA: poly-L-lactate; SC/BMSC: stem cells/bone marrow stem cells; SF: silk fibroin; TCP/βTCP: tricalcium phosphate.