Development&Evaluation Of A Hybrid Construction Made Of Hydroxyapatite/Nanocrystalline Silicon-substituted Hydroxyapatite (HA/nSiHA)&Demineralized/decellularized Bone Matrix(DBM/dDBM)

The objective of this novel interspecies study is to investigate the potential of a hybridconstruction consisting of DBM/dDBM produced from bovine, porcine, rat and humanbone and HA/nSiHA, to support the proliferation of rat’s bone marrow stem cells (BMSCs)and to provide a structure for the BMSCs differentiation into the osteoblast lineage. Theoverarching goal is to provide a preliminary in-vitro assessment of these constructions inorder to facilitate the development of an implantable hybrid construction with incorporatedosteoinductive and osteoconductive properties, appropriate for allogeneic transplantation/xenotransplantation in orthopaedic applications. A full-factorial design has been utilized toinvestigate the effect of the following factors: interspecies biocompatibility,decellularization intense, PH changes, variations of the DBM/dDBM powder with theweight ratio of polymers, absence of differentiation cell culture supplement and presence ofnSi. DBM/dDBM fragments were produced following a combination of protocols and2types/levels of decellularization samples were created (trypsin for24and48h) in order tosatisfy even more the intent of decellularization, reaching lower quantities of dsDNA than theminimal decellularization criteria of the50ng dsDNA/mg dDBM. HA/nSiHA powder wasproduced by the precipitation method. SEM, EDX, FT-IR, histological andimmunohistochemical stainings were used to characterize the materials. Five differentsubstrate mixtures (w/w%) were constructed;100%DBM/dDBM,40%DBM/dDBM:60%HA/nSiHA,20%DBM/dDBM:80%HA/nSiHA,10%DBM/dDBM:90%HA/nSiHA and100%HA/nSiHA. Non-porous discs/scaffolds with the above mixtures were constructedmanually and PH changes and degradation rates were measured after exposed in distilledwater and PBS for21and28days respectively. Cells were cultured on representativescaffolds and cytotoxicity and osteogenic differentiation were investigated. For thecytotoxicity experiment, cells were cultured for24h with mesenchymal stem cell growthmedium and analyzed using a cytotoxicity kit and SEM. The viability of the cells wasinversely related with the amount of the DBM/dDBM loading on the scaffolds and thehighest cell number in between the hybrid scaffolds (lower cytotoxicity) was observed inthe substrate mixture%w/w of10%DBM/dDBM:90%HA/nSiHA. For the differentiation experiment, cells shared in2groups and cultured respectively with and withoutdifferentiation cell culture supplement. The osteogenic differentiation was assessed withmorphological observations of the cells on the18thday as well with an alkaline phosphataseactivity (ALP) assay in two time points, days7and14. Interestingly, bovine, porcine andhuman hybrid constructions appeared with biocompatibility in their cell-substrateinteractions with the rat BMSCs. This study indicates that in vitro osteogenicdifferentiation was maintained even in between different species, even in the absence ofdifferentiation cell culture supplement. Thus differentiation may occur due to stimulationfrom the osteoinductive factors in the DBM/dDBM in interspecies cell-substrateinteractions. Interestingly, the hybrid groups cultured without the differentiation cell culturesupplement appeared similar osteogenic differentiation with the groups cultured with thedifferentiation cell culture supplement and higher osteogenic differentiation than the groupof100%nSiHA that cultured with differentiation culture supplement. Interestingly, thegroup of100%human DBM/dDBM appeared with one of the highest values in between allthe groups. The incorporation of nSi in the scaffold appeared with significant impact in themineralization process. The increased local pH, as well the chromatin remodeling of theresidual cellular content of the DBM/dDBM appeared to result in an environment that wasconducive for osteogenic differentiation. Therefore, the correlation between all theevaluation results indicates that in vitro osteoinductivity is possible due to interspeciescell-substrate interactions in the absence of differentiation culture supplement and isrespective to the mineral and cellular content of the DBM/dDBM. Subsequently, theoptimization of the demineralization/decellularization processes may lead in scaffoldingproperties tailored to suit specific orthopaedic applications in the fields of allogeneictransplantation/xenotransplantation.