Table 1.

Studies on CS-based scaffolds for bone tissue regeneration.

Authors	Scaffold type	Study outline	Results	Conclusion
Miranda et al. 2011 [8]	CS (DA: 15%; MW: NA) +GEL (3 : 1 ratio) as membranes (2D) or sponges (3D) crosslinked with glutaraldehyde	In 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): n = 5	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: n = 12	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 weeks	CS microspheres demonstrated great potential for use as BMP-2 matrix carrier for bone regeneration P < 0.01 (in vitro)
Costa-Pinto et al. 2012 [43]	CS (DA: NA; MW: NA) combined with PBS (1 : 1 ratio), with or without SC	In 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: n = 6	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: n = 23	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 (P < 0.05)
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: n = 10; n = 3 (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 control	CS+nHA has potential for use in bone regeneration of critical defects, favoring new bone formation when compared to CS alone (P < 0.05)
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: n = 3	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 CS	There was greater alveolar bone maturation after extraction with the use of CS+GEL+SC (P < 0.05)
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: n = 3	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 control	CS+ALG were biocompatible and permitted osteogenic growth and SC differentiation In vitro and presented osteoconductive properties in vivo (P < 0.05)
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 n = 3	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 (P < 0.05)
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 (P < 0.05), 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: n = 6 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: n = 4	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: n = 4	In vivo: greater bone formation in the CS+HA+BMP-2+SC group, with greater expression of collagen and osteocalcin, compared to blank scaffolds	CS+BMP2+CS demonstrated great potential for the regeneration of bone defects, with a synergistic effect of the combination (P < 0.05)
Koç et al. 2014 [53]	CS sponges (MW 400 kDa; DA < 15%) + HA (ratio: 9 : 1), whether or not activated with VEGF	In 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: n = 6	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 (P < 0.05)
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 (P < 0.05) 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-imides	In 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 (P < 0.05)
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: n = 2	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: n = 4	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 (P < 0.01)
Dhivya et al. 2015 [58]	Hydrogels of CS-Zn (DA: NA; MW: NA)+β-glycerophosphate + nHA (ratio: 8 : 1; 1) or without nHA	In 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 (P < 0.05)
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: n = 2 (control n = 3)	CS + copper exhibited greater bone neoformation than pure CS or control, both via micro-CT and via histological analyses	CS + copper has great potential for application in bone regeneration and promoted bone regeneration in vivo (P < 0.05)
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: n = 12	In vitro: scaffolds with spherical nHA demonstrated greater osteogenic proliferation and differentiation (P < 0.01)   In vivo: CS+GEL+ nHA+SC showed greater bone-like tissue formation than acellular scaffolds (P < 0.01); spherical nHA induced thicker bone-like formation after 12 weeks (P < 0.05)	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: n = 3	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 (P < 0.05) 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+SC	In 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: n = 2 (histology); n = 6 (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 (P < 0.01), 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.