A hydrogel based on chitosan, collagen, hydroxypropyl-γ-cyclodextrin and polyethylene glycol was developed and characterized. The incorporation of nano-hydroxyapatite and pre-encapsulated hydrophobic/hydrophilic model drugs diminished the porosity of hydrogel from 81.62 ± 2.25% to 69.98 ± 3.07%. Interactions between components of hydrogel, demonstrated by FTIR spectroscopy and rheology, generated a network that was able to trap bioactive components and delay the burst delivery. The thixotropic behavior of hydrogel provided adaptability to facilitate its implantation in a minimally invasive way. Release profiles from microspheres included or not in hydrogel revealed a two-phase behavior with a burst- and a controlled-release period. The same release rate for microspheres included or not in the hydrogel in the controlled-release period demonstrated that mass transfer process was controlled by internal diffusion. Effective diffusion coefficients,
D
eff
, that describe internal diffusion inside microspheres, and mass transfer coefficients,
h
, i.e. the contribution of hydrogel to mass transfer, were determined using ‘genetic algorithms’, obtaining values between 2.64·10
−15
and 6.67·10
−15
m
2
/s for
D
eff
and 8.50·10
−10
to 3.04·10
−9
m/s for
h
. The proposed model fits experimental data, obtaining an
R
2
-value ranged between 95.41 and 98.87%.
In vitro
culture of mesenchymal stem cells in hydrogel showed no manifestations of intolerance or toxicity, observing an intense proliferation of the cells after 7 days, being most of the scaffold surface occupied by living cells.