Table 4: Studies on CS-based scaffolds for cartilage tissue regeneration.

AuthorsScaffold typeStudy outlineResultsConclusion

Chen et al. 2011 [80]CS sponges (DA 15%; 
MW 400 kDa) +HA+GEL (ratio NA) whether or not activated by growth factors (BMP-2 and TGF)
In vitro: CC and cell differentiation in MSC culture  
In vivo: implantation of sponges in osteochondral patellar defects of 4-month-old New Zealand white rabbits; HT and IHC evaluations  
Control group: DNA-free composite osteochondral graft  
Sample:
Control:
Growth and osteochondral differentiation in vitro were observed  
In vivo: greater osteochondral tissue neoformation with the scaffolds groups, with or without growth factors when compared to the control
CS sponges + HA+GEL with TGF and BMP-2 promoted greater cell growth and bone and cartilage tissue regeneration ()

Whu et al. 2013 [81]CS (MW 65 kDa; DA 40%) +GEL (ratios 5 : 0, 4 : 1, 3 : 2, 1 : 1, 2 : 3, 1 : 4, or 0 : 5) in films and sponges, either crosslinked or not with carbodiimideIn vitro: CC in culture of chondrocytes with scaffolds  
In vivo: implantation of crosslinked scaffolds in cartilage defects in rabbits feet; HT and IHC analyses  
Control group: untreated defects 
Sample:
In vitro: greater cell proliferation and viability with crosslinked CS+GEL scaffolds  
In vivo: greater regeneration of cartilage with CS+GEL impregnated with chondrocytes after 1 month There was no comparison with pure CS or with absence of chondrocytes
The authors conclude that carbodiimide crosslinked CS+GEL scaffold demonstrated potential for cartilage regeneration ()

Zhang et al. 2013 [82]Sponges of CS (MW 40 kDa; DA: NA) +PLGA (ratio 1 : 1), either with or without incorporation of SCIn vitro: CC in adipose-derived stem-cell culture, in chondrogenic medium  
In vivo: implantation of scaffolds, with or without SC, in articular defects in 4-month-old New Zealand rabbits knees; HT, IHC, and biomechanical assays (compressive modulus and cytonano-indentation)  
Control group: scaffold alone  
Sample:
In vitro: CS+PLGA favored chondrogenic adhesion, proliferation, and differentiation  
In vivo: CS+PLGA+SC promoted greater regeneration of the defects and maintenance of subchondral bone, after 12 weeks, and greater mechanical performance than scaffolds without SC
CS+PLGA+SC scaffolds were capable of regenerating the full thickness of the cartilage defects in 12 weeks ()

Deng et al. 2013 [83]Sponges of CS (DD: NA; MW: NA) +SF (ratio 1 : 1); DA: NA; MW: NA incorporated or not with SCIn vitro: CC in BMMSC culture  
In vivo: filling of defects in the cartilage of 2-3- month-old New Zealand rabbit knees with scaffolds, with or without SC; HT and IHC analyses  
Control group: untreated group 
Sample:
In vitro: CS scaffolds promoted chondrogenic differentiation  
In vivo: the CS+SF+SC scaffold promoted almost complete repair of the defects and positive HT and IHC results; CS+SF scaffold demonstrated better results than the control, but not as good as in the group with SC
CS+SF scaffold showed itself to be effective as SC carrier and capable of being used in the regeneration of cartilage tissue ()

Wu et al. 2014 [84]Sponges of pure CS (DA: NA; MW: NA) or combined with fibrin (ratio NA), whether or not incorporated with SCIn vitro: CC and chondrogenic differentiation in SC culture from synovial fluid  
In vivo: implantation in defects in 2-week-old nude mice TMJ disc; HT, IHC, and PCR analyses Control group: cell-free chitosan/fibrin scaffold  
Sample:
In vitro: CS + fibrin exhibited greater chondrogenic adhesion, proliferation, and differentiation  
In vivo: CS+SC and CS+fibrin+SC induced greater regeneration than acellular scaffolds at 4 weeks
CS + fibrin scaffold with TMJ-derived stem cells demonstrated regenerative capacity for the treatment of TMJ disc perforations ()

Cheng et al. 2014 [32]Membranes of CS (DA ≤ 10%; MW: 200–500 kDa) +PLGA (ratio 75 : 25), whether or not impregnated with chondrocytesIn vitro: CC and chondrogenic differentiation inBMMSC culture  
In vivo: implantation in cartilage defects of 2-month-old New Zealand rabbit ears; macroscopic and HT analyses  
Control group: untreated defects 
Sample:
CS+PLGA + chondrocytes demonstrated complete and homogeneous regeneration after 18 weeks, with the formation of mature cartilage tissue; acellular scaffold and control group exhibited fibrosisCS+PLGA impregnated with chondrocytes were capable of regenerating the cartilage tissue ( not reported)

Ravanetti et al. 2015 [85]CS+ raffinose (DA: NA; MW: NA; ratio: NA)In vivo: implantation of scaffolds in osteochondral defects in the scapula of New Zealand white rabbits; macroscopic and HT analyses; negative control  
Control group: untreated defects  
Sample:
CS + raffinose did not promote regeneration of defects histologically or macroscopically and induced inflammation and formation of fibrous capsule, after 4 weeksThe authors conclude that CS + raffinose has limitations and that further studies are needed before application.

Ravindran et al. 2015 [86]CS+COL (1 : 1), with or without SC and ECM (DA: NA; MW: NA)In vitro: culture of MSC in osteogenic and chondrogenic medium 
In vivo: implantation in the subcutaneous tissue of mice; HT, IHC, and magnetic resonance analyses  
Control group: scaffolds without ECM  
Sample: not specified
In vitro:ECM scaffolds induced chondrogenic differentiation 
In vivo: CS+COL+SC+ECM presented expression of chondrogenic differentiation markers after 2 weeks; with magnetic resonance, newly formed tissue similar to native cartilage was observed after 8 weeks
The CS+COL+SC+ECM scaffold demonstrated efficiency in the regeneration of cartilage and bone tissue

Meng et al. 2015 [87]CS hydrogel, either with or without DBM particles, E7 peptide (P7), and SC (DA = NA; MW = NA; ratio = NA)In vitro: CC and chondrogenic differentiation in culture of BMSC; compression strength and elastic modulus tests  
In vivo: HT and IHC analyses of subcutaneous tissue of nude mice after 4 weeks  
Control group: pure CS scaffolds and composite scaffolds of DBM and CS;  
Sample:
In vitro: greater cell proliferation and differentiation with CS+DBM+P7 Preparation of DBM particles might influence the mechanical properties of scaffolds and cell proliferation  
In vivo: CS+DBM+P7+SC produced greater cartilage tissue formation than pure CS or with DBM; no negative control
CS+DBM+P7 hydrogel combined with mesenchymal stem cells has potential for regeneration of cartilage tissue.

Zhang et al. 2015 [88]CS sponges (MW 40 kDa; DA < 5%) +PLGA, with or without SC; ratio: NA; average viscosityIn vitro: CC and chondrogenic differentiation in culture of SC  
In vivo: implantation in cartilage defects in 4-month-old New Zealand rabbit knees; macroscopic, HT and IHC analyses; negative control not specified  
Control group: adherent ASC/scaffold complexes  
Sample:
In vitro: CS+PLGA+SC demonstrated chondrogenic differentiation  
In vivo: the scaffold promoted new formation of cartilage similar to hyaline, both histologically and via biomechanical evaluation, after 6 and 12 weeks
CS+PLGA sponges incorporated with aggregated stem cells represents a promising technique in tissue regeneration

CC: cytocompatibility; COL: collagen; CS: chitosan; DA: degree of acetylation; DBM: Demineralized bone matrix; ECM: extracellular matrix; HT: histological; IHC: immunohistochemical; kDa: kilodaltons; MW: molecular weight; PCR: polymerase chain reaction; PLGA: polylactic-co-glycolic acid; SC/BMSC: stem cells/bone marrow stem cells; TMJ: temporomandibular joint.