New injectable two-step forming hydrogel for delivery of bioactive substances in tissue regeneration

Regenerative Biomaterials, 2019, 149–162 doi: 10.1093/rb/rbz018 Advance Access Publication Date: 10 May 2019 Research Article New injectable two-step forming hydrogel for delivery of bioactive substances in tissue regeneration 1,2 and Edgar Pérez-Herrero 1,2,†, Patricia Garcı́a-Garcı́a1,†, Jaime Gómez-Morales3, Matias Llabrés1,4, Araceli Delgado Carmen Évora 1,2,* 1 Department of Chemical Engineering and Pharmaceutical Technology, University of La Laguna, La Laguna 38200, Tenerife, Spain; 2Institute of Biomedical Technologies (ITB), Center for Biomedical Research of the Canary Islands (CIBICAN), University of La Laguna, La Laguna 38071, Tenerife, Spain; 3Laboratory of Crystallographic Studies, Andalusian Earth Sciences Institute, Spanish Research Council—University of Granada, Armilla, Granada 18100, Spain; 4Institute of Tropical Diseases and Healthcare of the Canary Islands, University of La Laguna, La Laguna 38203, Tenerife, Spain *Correspondence address. Department of Chemical Engineering and Pharmaceutical Technology, University of La Laguna, La Laguna 38200, Tenerife, Spain. E-mail: † These authors have contributed equally in this work. Received 20 December 2018; revised 6 March 2019; accepted on 21 March 2019 Abstract A hydrogel based on chitosan, collagen, hydroxypropyl-c-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 6 2.25% to 69.98 6 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, Deff, 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 2.641015 and 6.671015 m2/s for Deff and 8.501010 to 3.04109 m/s for h. The proposed model fits experimental data, obtaining an R2-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. Keywords: hydrogel; collagen–cyclodextrin–chitosan; rheology; mass transfer; estradiol; FITC-dextran Introduction Hydrogels are three-dimensional hydrophilic polymeric networks, which are able to absorb and retain large quantities of water, solvents or biological fluids in the free space of their structure without being dissolved in the same media. They can be generated through physical crosslinking by weak cohesive forces, like ionic or hydrogen bonds and p–p or van der Waals interactions, or chemical crosslinking by stable covalent forces that improve the mechanical properties C The Author(s) 2019. Published by Oxford University Press. V This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 149 150 The toxicity of these compounds decreases with increasing number of glucopyranose units, thus, gamma cyclodextrins are less toxic than alpha or beta ones [11]. Synthetic polymers, like poly(DL-lactide-co-glycolide) (PLGA), poly(DL-lactide) (PLA), poly(vinyl alcohol) (PVA), poly(acrylic acid) (PAA), polyethylene glycol (PEG), can enhance and control the mechanical strength and degradation rates of hydrogels. Still, they do not provide the optimal environment for cell inclusion and tissue regeneration, so, a combination of both types of polymers, natural and synthetic, must be used for the biomedical application of hydrogels [5, 12, 13]. Moreover, the incorporation of inorganic components into the hydrogel structure, such as hydroxyapatite (HAp) i.e. part of the bone matrix, have been used in hard tissue repair to reinforce the mechanical properties of the hydrogel [14]. In this regard, nanosized HAp (nano-HAp) should be used to promote their degradation by osteoblastic enzymes, like the alkaline phosphatase, and form new tissue by osteoblasts, being this size the optimum for enhanced cell adhesion and proliferation [14]. Bioactive substances can be released from hydrogels by either degradation of polymer or diffusion through the pores of their structure, or by a combination of both. Release profiles in these systems typically reveal a two-phase behavior, a burst step followed by a slow controlled release period. Because of burst may cause high concentrations of bioactive substances in the application site and consequently loss of these drugs, more complex systems are required to reduce such burst profiles, maintaining the total delivery values in longer periods. In this work, an innovative hydrogel based on biocompatible and biodegradable polymers, such as, chitosan, collagen, hydroxypropyl-c-cyclodextrin (HP-c-CD) and PEG, has been designed, developed and characterized in terms of porosity, rheology and mass transfer studies. The hydrogel was injectable, adaptable to treatment sites with diverse dimensions and shapes, easily crosslinked by means of TPP and blue light and presented adequate characteristic for good cell adhesion, viability and proliferation. Moreover, 17-bestradiol or rhodamine-B-isothiocyanate-dextran (RITC-dextran), as low molecular weight hydrophobic or high molecular weight hydrophilic model drugs, respectively, were encapsulated in PLGA/ PLA microspheres and included in the hydrogel, together with the nano-HAp for further use in bone regeneration. Materials and methods Materials R PLGA ResomerV RG504 (acid-terminated, lactide:glycolide 50:50, Mw 38–54 kDa) and RG858S (ester-terminated, lactide:glycolide 85:15, Mw 190–240 kDa) and PLA ResomerV RG203-S (ester-terminated, Mw 18–28 kDa) were acquired from Boehringer-Ingelheim (Germany). HP-c-CD (CAVASOLV W8 HP) was obtained from Wacker (Germany). Ultrapure chitosan ProtasanTM UP-CL-213 (86% deacetylation, viscosity 150 mPas at 1 wt. % aqueous solution) and ultrapure alginate PronovaTM UP MVG were purchased from NovaMatrix (Norway). Bovine collagen, type I, was purchased from CellSystems Biotechnologie Vertrieb GmbH (Germany). Calcium chloride dihydrate (Bioxtra, 99% pure), sodium citrate triba (...truncated)