Effects Of Intrafibrillar-silicified Collagen Scaffolds On The Osteogenic Differentiation Of Human Dental Pulp Stem Cells

Bone loss, caused by tumors, trauma and arthritis, has been seriously affectinghuman’s health and life quality. Although autogenous bone grafts are regarded as the bestchoice for bone restoration, their major disadvantages include limited autogenous bonesource, additional surgical trauma, complications at the donor site, and long operationduration. Allogenic and xenogeneic bone grafts are greatly limited in clinical applicationbecause of their potential to transmit viral infections, poor biocompatibility andimmunogenicity toward human tissues. Therefore, the manufacture of the ideal bonesubstitute has become the critical point of in the repair of bone defect. With the rapiddevelopment of tissue engineering, stem-cell-based bone tissue engineering has becomeone of the hotspots in the research of bone regeneration, and a promising approach tosettle these problems.To successfully regenerate the functionalized bone, stem cells, scaffolds, and bioactive molecules, which are the critical components of bone tissue engineering, must becarefully selected and integrated appropriately. Although bone marrow-derivedmesenchymal stem cells (BMSCs) have good osteogenic differentiation potential and havebeen commonly used for bone regeneration, they are difficult to achieve a breakthrough inthe clinical application because the process to obtain BMSCs is difficult and the patientshave to suffer relatively extensive trauma and high expense. Compared with BMSCs, thesurgical access to obtain human dental pulp stem cells (hDPSCs) is relative easy andnon-traumatic without any economic burden for patients, and the clone forming ability ofhDPSCs is higher. Moreover, previous studies have indicated that hDPSCs candifferentiate into osteoblasts and form bone-like tissues. Thus, hDPSCs may replaceBMSCs as autologous seed cells in bone tissue engineering.Metals, ceramics, natural polymers and synthetic polymers have all been used asscaffolds in bone tissue engineering, but none of these materials could meet therequirements of ideal tissue engineering scaffolds. Although type I collagen is the mostabundant extracellular protein in bone and the frame for bone formation, and has beenfrequently used as natural scaffolds in bone regeneration, the poor mechanical propertiesand uncontrollable fast degradation rate have greatly limited its application. To addressthese problems, we have previously created intrafibrillar-silicified collagen scaffolds(ISCS) by incorporating choline-stabilized silicic acid into highly-porous type I collagenscaffolds. The ISCS significantly improve the mechanical properties and the resistance toprotease degradation of nonsilicified collagen scaffolds (NCS). In addition, ISCS arebiocompatible, and could promote the proliferation and osteogenic differentiation ofBMSCs.Although hDPSCs and BMSCs are similar in many aspects, and we have shown thatISCS could significantly stimulate the osteogenic differentiation of BMSCs in ourprevious studies, it is still not known whether ISCS have similarly osteoinductivefunctions on hDPSCs. Thus, the objective of the present study was to investigate thepotential use of hDPSCs and ISCS in bone tissue engineering, and the effects of ISCS onthe proliferation, osteogenic differentiation, and mineralization of hDPSCs. The main contents of our research are summarized as follows:1. Effects of ISCS on the cell viability of hDPSCsHuman DPSCs were separated from human dental pulp and identified throughcolony-forming efficiency analysis and cell surface marker analysis. The biocompatibilityof ISCS was investigated through observation of the cell shape and cell apoptosis analysis.Cell cycle analysis and DNA content assay were used to investigate the effects of ISCS onthe proliferation of hDPSCs. Our results indicated that the clone formation rate of hDPSCswas25%, and the shape of most hDPSCs was spindle. Results of cell surface markeranalysis showed high expressions of mesenchymal surface markers and low expressions ofhematopoietic surface makers. No significant difference could be detected among thepercentage distributions of normal cells in the ISCS group, NCS group and blank controlgroup (p＞0.05). The S-phase cell fraction (SPF) and DNA content of hDPSCs exposed toISCS were significantly higher than those of the NCS group or the blank control group (p＜0.05).2. Effects of ISCS on the osteogenic differentiation of hDPSCs in vitroIn the second part of our research, we investigated the effects of ISCS on theosteogenic differentiation and mineralization of hDPSCs in vitro, through quantitativereverse transcription–polymerase chain reaction (qRT-PCR), western blot, alkalinephosphatase (ALP) staining, ALP activity analysis, alizarin red S staining and vonkossastaining. Results of qRT-PCR and western blot showed that the hDPSCs exposed to ISCSshowed the highest expression levels of the osteogenic marker genes and proteins amongthe ISCS group, NCS group and negative control group (p＜0.05). Compared with NCS,ISCS could significantly promote the ALP activity and calcium deposits of hDPSCs (p＜0.05).3. Effects of ISCS on the osteogenic differentiation of hDPSCs in vivoThe cell-scaffold constructs were manufactured through seeding hDPSCs onto ISCSand NCS, respectively. Then the cell-scaffold constructs were implanted subcutaneously innude mice and harvested after8weeks. The samples were processed for alizarin red Sstaining, hematoxylin-eosin(HE) staining, and immunohistochemical staining for osteocalcin (OCN). Compared with the NCS group, much more osteocyte-like cells andcapillaries containing red blood cells could be identified within the newly formed bonetrabeculae in the ISCS group. ISCS could promote the calcium deposits and the expressionlevels of OCN more significantly than NCS (p＜0.05).Taken together, our results have indicated that ISCS could significantly stimulate theproliferation, mineralization and osteogenic differentiation of hDPSCs, when comparedwith the use of conventional NCS. The potential use of hDPSCs and ISCS to promotebone regeneration offers a promising approach for clinical bone repair and regeneration.