Preparation And Properties Of Injectable Hydrogels Derived From Demineralized And Decellularized Bone Extracellular Matrix

Objectives The purposes of this present study were to apply a stringent delipidation,demineralization and decellularization process to DBM prepared from porcine bone and resulting ECM materials.Gelation was induced under certain conditions.We characterize the structure and composition of physical and chemical characteristics of the ECM gels.It describes with characterization of the gelation kinetics,rheological properties and in vitro cytocompatibility of the gels.The present study provides an experimental basis for bone tissue engineering to provide an injectable ECM hydrogel implant material.Methods Fresh porcine femurs were harvested from pig aged 12–24 months.The cancellous were received in segmented form from fresh porcine femurs.The segments were cleaned of residual tissue and blood,dried at room temperature.Washed segments were sectioned to produce fragments no greater than 5 mm ×5 mm× 5 mm in commercial milk machine.The lipid in the segments was then extracted 48 h with a 1:1 mixture of chloroform and methanol at 4 °C and replaced the liquid once every 24 hours,then repeatedly rinsed and dried at room temperature.The segments were demineralized under agitation in 0.6 N HCL(30ml/g bone)at 4 °C for 48 h,and replaced the liquid once every 24 hours.The beaker was then set on a magnetic stir plate with the stir speed adjusted to yield a small vortex.The particles were suspended in motion in the acid and did not settle during the process.The particles were repeatedly rinsed and dried at room temperature,and decellularized in a solution of 1% Triton X-100 at 4 °C 24 h,then was rinsed with distilled water and dried at room temperature.The segments were dealt with DNase I and RNase mixed solution for 12 hours,then washed in distilled water and stored at-20 °C overnight.Frozen granules were freeze-dried in freeze dryer for 24 hours,resulting extracellular matrix ECM,stored at-20 °C until required after Co60 sterilization.Lyophilized ECM were added to 1 mg/ml pepsin in 0.01 N HCL for a final concentration of 10 mg matrix per ml suspension,i.e.1 g dry matrix was mixed with 100 mg pepsin in 10 ml of 0.01 N HCL.The suspension was mixed on a stirrer plate at 4 °C for 48 h,until no visible pieces of matrix remained.The resultant ECM digests were stored at-20 °C until required.Gelation was induced by neutralizing the salt concentration and p H of the pepsin digests solution at 4 °C followed by warming to 37 °C.We gave a final concentration of 4,6,8,10 mg/ml,respectively.The p H values were detected by a p H acidimeter at 4?.The gelation time was detected in incubator at 37?.Hydrogel morphology was made by naked eye view.Hydrogel were assessed by toluidine blue staining and type I collagen immunohistochemical methods and Safranin O staining.Content of GAG,collagen and DNA were measured.Biomechanics for compressive properties was assessed.The sterility of the produced materials was achieved by exposing bone samples to a 25 k Gy dose of gamma irradiation.In vitro biocompatibility of the hydrogel was assessed by Human umbilical cord MSCs seeded on hydrogels.Cytotoxicity test and cell proliferation was detected by CCK-8 assay.Live/dead assay was used to visualize cell morphology.The osteoinduction of the hydrogel was assessed by ALP assay and von Kossa staining of human umbilical cord MSCs seeded on hydrogels.The hydrogel was subcutaneously implanted in mice,and in vivo biocompatibility of the hydrogel was observed by naked eye view and HE staining.Results The gelation time at 37?ranged from 30 to 60 min.Hydrogel generally was crystal clear,smooth and moist surface,rich in water and with flexibility,which can quickly rebound after metal stripping pressure.HE staining and scanning electron microscopy showed the hydrogel had porous network structure which was irregularly shaped.HE showed that no cell nucleus and cell membrane related residues.Toluidine blue stains,type I collagen immunohistochemical methods and Safranin O staining showed positive.Hydrogel freeze-dried material show that the pore size is300.1 ± 38.12?m.ECM hydrogel water absorption of 975 ± 85%.Collagen content 0.93±0.06mg/mg dry weight,GAG content 8.78±0.12?g/mg dry weight,double stranded DNA content 1.3±0.3ng/mg dry weight,double stranded DNA(ds DNA)per mg initial dry weight <50ng.The storage modulus of the 10 mg/ml ECM hydrogel 300±21Pa.Turbidmetric gelation kinetics of ECM hydrogels showed sigmoidal shapes.CCK-8 assay and Live/dead assay showed no significant effect on cell proliferation and no toxicity.ALP assay,Van Gieson staining solution and alizarin red staining was positive,which suggested that human umbilical cord MSCs differentiated into osteoblasts.The in vivo experiments showed gelation in situ after injection,and there was no significant inflammatory response in the injection site.2 weeks after inoculation,the hydrogel was partially absorbed,with no obvious inflammatory cells infiltration or foreign body reaction.Conclusion We sought to produce a soluble form of the ECM materials which could be induced to polymerize into a gel.The long-term objective of this work was to develop a gel form of ECM biological material derived from porcine cancellous bone that retains osteoconductivity and osteoinductivity.ECM hydrogels characterized by lower storage modulus.ECM hydrogel showed low immunogenicity and better biocompatibility.It may be a new kind of ideal bone substitute and will be applied widely in the future.Decellularized tissue is increasingly frequent in regenerative medicine and tissue engineering strategies,a promising approach to repair or regenerate the bone tissue.