A novel bioactive implant enriched with PDLCs and EPCs enhances osseointegration in irradiated bone

Abstract

The study aimed to enhance early osseointegration in irradiated rabbit bone by co-culturing endothelial progenitor cells (EPCs) and periodontal ligament cells (PDLCs) on porous tantalum trabecular metal (PTTM) implants. 1) PDLCs were isolated from the periodontal ligament of sound rabbit premolars by enzymatic digestion method. Multidifferentiation potential, fluorescence assorted cell sorting (FACS), and immunofluorescence (STRO-1, CD146, CD90, vimentin, desmin, CD45, and CD34) were used for the characterization of PDLCs. 2) EPCs were isolated from peripheral blood by density centrifugation followed by double sorting with FACS (CD34CD133). CD34+CD133+EPCs were characterized by functional assays, FACS, and immunofluorescence (CD34, CD133, CD31, VEGFR-2, CD14, and CD45). 3) The isolated and characterized PDLCs and CD34+CD133+EPCs were cultured on PTTM disc (3.75´1mm) according to the four groups {I [Implant only], IP [Implant+PDLCs], IE [Implant+EPCs], and IPE [Implant+PDLCs (1)+EPCs (1)]} and evaluated for the proliferative potential, cell viability, adhesion, and penetration by CCK-8, live/dead staining, scanning electron microscopy (SEM) and backscatter SEM (BSEEM) respectively. 4) Left or right distal femoral condyles of 16 rabbits (Age > 8 months) were randomly irradiated (4 × 5cm) by a single dose of 15 Gy, whereas the contralateral leg served as a control. One week after irradiation, the cell-implant (CelPlant) complex was placed in both irradiated and normal condyles of each rabbit. 32 implants (4.1´10 mm) were placed in four groups {I [Implant only], IP [Implant+PDLCs], IE [Implant+EPCs], and IPE [Implant+PDLCs(1)+EPCs(1)]}. Rabbits were euthanized after three weeks. Effects of CelPlants on osseointegration were measured using implant stability quotient (ISQ), micro-CT, sequential fluorochrome labeling (SFL), BSSEM and histologically by Goldner's trichrome. Paired t-test, one-way ANOVA, 2-way ANOVA, independent sample t-test, and post-hoc Tukey HSD tests were used for statistical analysis. PDLCs (8/10) and EPCs (7/25) were successfully isolated and displayed their characteristic osteogenic (3/3) and angiogenic potential (4/4) respectively. PDLCs or EPCs revealed satisfactory proliferation, viability, attachment, and penetration on all discs. All rabbits completed in vivo study uneventfully. Significantly lower implant stability at baseline (ISQps) was found in all groups of irradiated sites (p<0.05) as compared to non-irradiated sites. No significant difference in ISQps was found among groups, but a significant increase in implant stability was found after 3 weeks (ISQss) among groups (p<0.05) in both irradiated and non-irradiated sites. In non-irradiated sites, there was no statistically significant difference (p>0.05) in ISQss between IPE and IE group, but both had significantly higher (p<0.05) ISQss than I group. However, in the irradiated samples, only IPE group had significantly higher (p<0.05) ISQss than I group. Similarly, both Micro-CT (BV/TV) and SFL demonstrated that bone formation was significantly higher (p<0.05) in the IPE and IE group of non-irradiated samples as compared to IP or I group. However, in irradiated samples, only IPE group had significantly higher (p<0.05) bone formation than all other groups. The above findings were also confirmed qualitatitively by BSSEM and histological analysis. Therefore, PDLCs and EPCs appeared to have synergistic effects on enhancing implant osseointegration in irradiated bone, whereas, in normal bone, EPCs alone might be sufficient to augment the osseointegration.