Degradation characteristics, cell viability and host tissue responses of PDLLA-based scaffold with PRGD and β-TCP nanoparticles incorporation

Abstract

This study is aimed to evaluate the degradation characteristics, cell viability and host tissue responses of PDLLA/PRGD/β-TCP (PRT) composite nerve scaffold, which was fabricated by poly(d, l-lactic acid) (PDLLA), RGD peptide(Gly-Arg-Gly-Asp-Tyr, GRGDY, abbreviated as RGD) modified poly-{(lactic acid)-co-[(glycolic acid)-alt-(l-lysine)]}(PRGD) and β-tricalcium phosphate (β-TCP). The scaffolds' in vitro degradation behaviors were investigated in detail by analysing changes in weight loss, pH and morphology. Then, the 3-(4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2 -H-tetrazolium bromide (MTT) assay and cell live/dead assay were carried out to assess their cell viability. Moreover, in vivo degradation patterns and host inflammation responses were monitored by subcutaneous implantation of PRT scaffold in rats. Our data showed that, among the tested scaffolds, the PRT scaffold had the best buffering capacity (pH = 6.1-6.3) and fastest degradation rate (12.4%, 8 weeks) during in vitro study, which was contributed by the incorporation of β-TCP nanoparticles. After in vitro and in vivo degradation, the high porosity structure of PRT could be observed using scanning electron microscopy. Meanwhile, the PRT scaffold could significantly promote cell survival. In the PRT scaffold implantation region, less inflammatory cells (especially for neutrophil and lymphocyte) could be detected. These results indicated that the PRT composite scaffold had a good biodegradable property; it could improve cells survival and reduced the adverse host tissue inflammation responses.

Keywords: PDLLA/PRGD/β-TCP scaffold; cell viability; degradation; host tissue responses.

Figure 1.

PH changes of P, PR, PT and PRT scaffolds degraded in vitro. (P: poly(d,l-lactic acid); PR: poly(d,l-lactic acid)/RGD peptide modification of poly{(lactic acid)-co-[(glycolic acid) -alt-(l-lysine)]}; PT: poly(d,l-lactic acid)/β-tricalcium phosphate; PRT: poly(d,l-lactic acid)/RGD peptide modification of poly{(lactic acid)-co-[(glycolic acid) -alt-(l-lysine)]}/β-tricalcium phosphate).

Figure 2.

Weight loss ratio of P, PR, PT and PRT scaffolds degraded in vitro. (P: poly(d,l-lactic acid); PR: poly(d,l-lactic acid)/RGD peptide modification of poly{(lactic acid)-co-[(glycolic acid) -alt-(L-lysine)]}; PT: poly(d,l-lactic acid)/β-tricalcium phosphate; PRT: poly(d,l-lactic acid)/RGD peptide modification of poly{(lactic acid)-co-[(glycolic acid) -alt-(l-lysine)]}/β-tricalcium phosphate).

Figure 3.

Morphology of P, PR, PT and PRT scaffolds degraded in vitro. (Scale bars: 50 μm in A–D, 10 μm in E–H. P: poly(d,l-lactic acid); PR: poly(d,l-lactic acid)/RGD peptide modification of poly{(lactic acid)-co-[(glycolic acid) -alt-(l-lysine)]}; PT: poly(d,l-lactic acid)/β-tricalcium phosphate; PRT: poly(d,l-lactic acid)/RGD peptide modification of poly{(lactic acid)-co-[(glycolic acid) -alt-(l-lysine)]}/β-tricalcium phosphate).

Figure 4.

PC12 cell viability cultured in the degradation liquid of P, PR, PT and PRT scaffolds. (P: poly(d,l-lactic acid); PR: poly(d,l-lactic acid)/RGD peptide modification of poly{(lactic acid)-co-[(glycolic acid) -alt-(l-lysine)]}; PT: poly(d,l-lactic acid)/β-tricalcium phosphate; PRT: poly(d,l-lactic acid)/RGD peptide modification of poly{(lactic acid)-co-[(glycolic acid) -alt-(l-lysine)]}/β-tricalcium phosphate)

Figure 5.

PI/Hochst33342 staining (A) and cell death ratio (B) after cultured in the degradable solution of P, PR, PT and PRT scaffolds. (*P < 0.05, compared with P scaffold. P: poly(d,l-lactic acid); PR: poly(d,l-lactic acid)/RGD peptide modification of poly{(lactic acid)-co-[(glycolic acid) -alt-(l-lysine)]}; PT: poly(d,l-lactic acid)/β-tricalcium phosphate; PRT: poly(d,l-lactic acid)/RGD peptide modification of poly{(lactic acid)-co-[(glycolic acid) -alt-(l-lysine)]}/β-tricalcium phosphate)

Figure 6.

Morphology of P and PRT scaffolds degraded in vivo. (Scale bars: 50 μm in A and B, 10 μm in C and D. P: poly(d,l-lactic acid); PRT: poly(d,l-lactic acid)/RGD peptide modification of poly{(lactic acid)-co-[(glycolic acid) -alt-(l-lysine)]}/β-tricalcium phosphate)

Figure 7.

HE staining of subcutaneous tissue after P and PRT scaffolds implantation for 8 weeks. (Solid arrow: neutrophils; arrow head: lymphocytes; dotted arrow: monocytes. (A) and (C) Scale bar =50 μm; (B) and (D) Scale bar =20 μm. P: poly(d,l-lactic acid); PRT: poly(d,l-lactic acid)/RGD peptide modification of poly{(lactic acid)-co-[(glycolic acid) -alt-(l-lysine)]}/β-tricalcium phosphate).

Figure 8.

The number of inflammation cells after P and PRT scaffolds implantation for 8 weeks. (*P < 0.05, **P < 0.01. P: poly(d,l-lactic acid); PRT: poly(d,l-lactic acid)/RGD peptide modification of poly{(lactic acid)-co-[(glycolic acid) -alt-(l-lysine)]}/β-tricalcium phosphate).