Amide-linkage formed between ammonia plasma treated poly(D,L-lactide acid) scaffolds and bio-peptides: enhancement of cell adhesion and osteogenic differentiation in vitro

The surface characteristics of scaffolds for bone tissue engineering must support cell adhesion, migration, proliferation, and osteogenic differentiation. In the study, poly(D,L ‐lactide acid) (PDLLA) scaffolds were modified by combing ammonia (NH₃) plasma pretreatment with Gly‐Arg‐Gly‐Asp‐Ser (GRGDS)‐peptides coupling technologies. The x‐ray photoelectron spectroscopy (XPS) survey spectra showed the peak of N1s at the surface of NH₃ plasma pretreated PDLLA, which was further raised after GRGDS conjugation. Furthermore, N1s and C1s in the high‐resolution XPS spectra revealed the presence of ? C?N (imine), ? C? NH? (amine), and ? C?O? NH? (amide) groups. The GRGDS conjugation increased amide groups and decreased amine groups in the plasma‐treated PDLLA. Confocal microscope and high performance liquid chromatography verified the anchored peptides after the conjugation process. Bone marrow mesenchymal stem cells were co‐cultured with scaffolds. Fluorescent microscope and scanning electron microscope photographs revealed the best cell adhesion in NH₃ plasma pretreated and GRGDS conjugated scaffolds, and the least attachment in unmodified scaffolds. Real‐time PCR demonstrated that expression of osteogenesis‐related genes, such as osteocalcin, alkaline phosphatase, type I collagen, bone morphogenetic protein‐2 and osteopontin, was upregulated in the single NH₃ plasma treated and NH₃ plasma pretreated scaffolds following GRGDS conjugation. The results show that NH₃ plasma treatment promotes the conjugation of GRGDS peptides to the PDLLA scaffolds via the formation of amide linkage, and combination of NH₃ plasma treatment and peptides conjugation may enhance the cell adhesion and osteogenic differentiation in the PDLLA scaffolds. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 682–694, 2011.     

Purification and characterization of an extracellular poly(L-lactic acid) depolymerase from a soil isolate, Amycolatopsis sp. strain K104-1

Poly(L-lactic acid) (PLA)-degrading Amycolatopsis sp. strains K104-1 and K104-2 were isolated by screening 300 soil samples for the ability to form clear zones on the PLA-emulsified mineral agar plates. Both of the strains assimilated >90% of emulsified 0.1% (wt/vol) PLA within 8 days under aerobic conditions. A novel PLA depolymerase with a molecular weight of 24,000 was purified to homogeneity from the culture supernatant of strain K104-1. The purified enzyme degraded high-molecular-weight PLA in emulsion and in solid film, ultimately forming lactic acid. The optimum pH for the enzyme activity was 9.5, and the optimum temperature was 55 to 60 degrees C. The PLA depolymerase also degraded casein and fibrin but did not hydrolyze collagen type I, triolein, tributyrin, poly(beta-hydroxybutyrate), or poly(epsilon-caprolactone). The PLA-degrading and caseinolytic activities of the enzyme were inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride but were not significantly affected by soybean trypsin inhibitor, N-tosyl-L-lysyl chloromethyl ketone, N-tosyl-L-phenylalanyl chloromethyl ketone, and Streptomyces subtilisin inhibitor. Thus, Amycolatopsis sp. strain K104-1 excretes the unique PLA-degrading and fibrinolytic serine enzyme, utilizing extracellular polylactide as a sole carbon source.