Table 2: Studies on CS-based scaffolds for cutaneous tissue regeneration.

AuthorsScaffold typeStudy outlineResultsConclusion

Guo et al.
2011 [63]
Bilayer CS (DA 15–25%; MW: 100–170 kDa) +COL membranes (ratio NA) impregnated or not with TMC (DD 90%) and VEGF (plasmid-DNA encoded)In vitro: CC in HUVEC culture 
In vivo: implantation of membranes on burn skin lesions on the backs of guinea pigs. HT, PCR, and Western-blot analyses  
Control groups: blank scaffolds and CS/COL/TMC/pDNA without VEGF 
Sample: not specified
Greater cell viability and VEGF expression in scaffolds with TMC/pDNA-VEGF than the controls in vitro  
Greater angiogenesis, VEGF expression and better repair of wounds with TMC/pDNA-VEGF scaffolds in vivo
CS+COL impregnated with TMC and VEGF promoted angiogenesis and dermal regeneration (), showing potential for use in the regeneration of epithelial lesions

Tchemtchoua et al. 2011 [64]Films, sponges, and CS nanofibrils (DA 16% and MW 67 kDa)In vitro: CC in a culture of fibroblasts, keratinocytes, and endothelial cells  
In vivo: implantation in the subcutaneous tissue (back skin) of 10-week-old mice (biocompatibility) and in skin defects; HT evaluation  
Control: untreated defects 
Sample:
In vitro: greater adhesion, cell proliferation, and differentiation with nanofibrillar CS 
In vivo: greater biocompatibility and faster regeneration of wounds with nanofibrillar CS; CS sponges caused foreign body reaction
The authors conclude that the nanofibrillar form has advantages over the others, being more biocompatible and effective in regeneration of the skin

Sundaramurthi et al. 2012 [65]CS (DA 15%; MW: NA) in nanofibrils or films (crosslinked with glutaraldehyde), in combination with PVA and RSPO1 (50 ng)In vitro: CC in fibroblast cell culture; evaluation by RT-PCR  
In vivo: implantation on skin wounds on rats’ backs; macroscopic and HT evaluation  
Control groups: untreated wounds (negative), Bactigras® (positive)  
Sample:
In vitro: greater cell adhesion and proliferation in nanofibrillar CS+PVA group ()  
In vivo: complete macroscopic regeneration after 2 weeks and better histopathological results in the CS+PVA+RSPO1 group ()
CS+PVA demonstrated good results as carrier of the growth factor, constituting a biocompatible biomaterial with potential for application as a skin substitute

Veleirinho et al. 2012 [66]CS (medium MW; DA: NA) combined with the polymer PHBV (ratios: 2 : 3 and 1 : 4)In vitro: CC in a culture of fibroblast cells of mouse;   
In vivo: implantation of scaffolds and a commercial biomaterial as a control in skin wounds on the backs of 2-month-old rats; macroscopic and HT evaluation of regeneration.  
Sample: not specified
In vitro: cell viability and proliferation with CS+PHBV (1 : 4) similar to the control ()  
In vivo: greater organization and maturation of the epithelial tissue with CS+PHBV (1 : 4); lower occurrence of inflammatory infiltrate with CS+PHBV (2 : 3)
CS+PHBV has potential for promoting skin regeneration, with biocompatibility in vitro

Wang et al. 2013 [67]CS membranes (MW 100 to 171 kDa; DA 15%) + COL and PLGA (ratio: NA)In vivo: implantation of the scaffolds, with or without PLGA, on skin defects in backs of 2-month-old rats; macroscopic, HT, IHC, PCE analyses and tensile strength tests.  
Sample:
CS+COL+PLGA scaffolds demonstrated better healing and greater expression of IHC and PCR markers and higher mechanical performance ()CS+COL scaffolds reinforced with PLGA demonstrated acceleration of angiogenesis and better skin regeneration than CS+COL ()

Sarkar et al. 2013 [68]Crosslinked CS membranes (MW 71 kDa; DA < 10%) whether or not combined with COLIn vitro: CC in culture of fibroblasts and keratinocytes.  
In vivo: implantation in human skin defects ex vivo; HT analyses.  
Control groups: not specified  
Sample:
In vitro: CS+COL demonstrated better cell adhesion, proliferation, and viability  
In vivo: CS+COL promoted partial reepithelialization with migration after 14 days; pure CS did not promote regeneration
CS+COL scaffold promoted better regeneration of skin wounds than pure CS scaffolds ()

Zeinali et al. 2014 [24]CS membranes (medium MW; DA 15–25%) crosslinked with PHBV, with or without SC ()In vitro: CC in umbilical cord SC culture;  
In vivo: implantation in skin defects on 4–8-week-old rats’ backs; HT and IHC analyses. Control not specified.  
Sample:
In vitro: CS+PHBV showed greater cell proliferation and viability  
In vivo: greater regeneration of cutaneous tissue with CS+PHBV+SC 
Statistical analysis not performed
CS+PHBV added to stem cells was capable of regenerating full thickness skin defects in rats

Guo et al. 2014 [69]Bilayer CS (MW 100–170 kDa; DA 15–25%) +COL and silicone membranes 
(ratio: NA)
In vivo: implantation of scaffolds in excisional or burnt skin lesions in guinea pigs; HT, IHC and IF evaluations; c 
Control group: commercial bandage;  
sample:
CS+COL produced results inferior to the control in the regeneration of burn lesions () There was no significant difference with excisional lesions ()CS and collagen demonstrated effectiveness similar to the commercial product in the regeneration of skin damaged by excisional wounds

Revi et al. 2014 [29]CS sponges 
(MW 354 kDa; DA 14%) impregnated or not with keratinocytes and fibroblasts
In vivo: implantation of scaffolds or unspecified commercial product (positive control) in dorsal skin lesions of rabbits; HT and IHC analyses; no negative control.  
Sample:
CS scaffolds with or without cells exhibited slower complete reepithelization of lesions (28 days) than commercial product  
(14 days) ( compared to CS without cells and compared to CS with cells)
CS sponges combined with dermal cells showed potential for application in the regeneration of complete skin defects

Han et al. 2014 [27]CS sponges + GEL (ratio NA) incorporated with antibacterial drugs.  
MW and DA not reported. Drugs not reported
In vitro: CC in culture of skin fibroblasts); porosity, water absorption and biodegradation tests;  
In vivo: implantation of scaffolds in skin lesions on rabbits’ backs; HT analysis of biocompatibility; no negative control.  
Sample:
In vitro: CS+GEL demonstrated adequate CC  
In vivo: inflammatory infiltrate present within 15 days, being lower in sponges with antimicrobials; there was no lesion regeneration  
Statistical analysis was not performed
CS+GEL exhibited adequate physicochemical properties and cytocompatibility in vitro, but induced inflammation in vivo

Ahamed et al. 2015 [70]CS+ cellulose (ratio NA), incorporated with nanoparticles of silver, with or without gentamicin.  
MW and DA not reported
In vivo: implantation in skin lesions in the backs of Wistar rats; macroscopic, biochemical and planimetric analyses.  
Controls: sterile cotton gauze dipped with gentamicin or standard soframycin ointment.  
Sample:
Scaffolds with or without gentamicin did not exhibit any difference between one another but were better than controls The healing was complete after 25 days  
not reported
CS + cellulose was effective in the regeneration of skin wounds

Wang et al. 2016 [71]COL+CS (DA < 15%; MW 106–171 kDa) + PLGA + PUR (ratio NA)In vivo: implantation in skin lesions on the backs of 2-month-old Sprague-Dawley rats; SEM, HT and IHC analyses; tensile strength tests;  
Control groups; commercial membrane (COL + silicon);  
Sample:
COL+CS+PLGA+PUR showed greater expression of angiogenesis markers, better regeneration of cutaneous tissue wounds and better mechanical performance than commercial membrane used as controlCOL+CS+PLGA+PUR membranes promoted better regeneration of skin defects in comparison with commercial membrane ()

CC: cytocompatibility; COL: collagen; CS: chitosan; DA: degree of acetylation; HT: histological; IHC: immunohistochemical; kDa: kilodaltons; MW: molecular weight; PCE: polycaprolactone-polyethylene glycol polymer; PHBV: poly(3-hydroxybutyrate-co-3-hydroxyvalerate; PLGA: polylactic-co-glycolic acid; PUR: polyurethane; PVA: polyvinyl-alcohol; RT-PCR: real time-polymerase chain reaction; RSPO1: R-spondin-1 angiogenesis growth factor; TMC: trimethyl chitosan chloride; VEGF: vascular endothelial growth factor.