Differentiation of smooth muscle progenitor cells in peripheral blood and its application in tissue engineered blood vessels

Background: A major shortcoming in tissue engineered blood vessels (TEBVs) is the lack of healthy and easily attainable smooth muscle cells (SMCs). Smooth muscle progenitor cells (SPCs), especially from peripheral blood, may offer an alternative cell source for tissue engineering involving a less invasive harvesting technique. Methods: SPCs were isolated from 5-ml fresh rat peripheral blood by density-gradient centrifugation and cultured for 3 weeks in endothelial growth medium-2-MV (EGM-2-MV) medium containing platelet-derived growth factor-BB (PDGF BB). Before seeded on the synthesized scaffold, SPC-derived smooth muscle outgrowth cell (SOC) phenotypes were assessed by immuno-fluorescent staining, Western blot analysis, and reverse transcription polymerase chain reaction (RT-PCR). The cells were seeded onto the silk fibroin-modified poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (SF-PHBHHx) scaffolds by 6×104 cells/cm2 and cultured under the static con- dition for 3 weeks. The growth and proliferation of the seeded cells on the scaffold were analyzed by 3-(4,5-dimethylthiazol-2-yl)- diphenyltetrazolium bromide (MTT) assay, scanning electron microscope (SEM), and 4,6-diamidino-2-phenylindole (DAPI) staining. Results: SOCs displayed specific "hill and valley" morphology, expressed the specific markers of the SMC lineage: smooth muscle (SM) α-actin, calponin and smooth muscle myosin heavy chain (SM MHC) at protein and messenger ribonucleic acid (mRNA) levels. RT-PCR results demonstrate that SOCs also expressed smooth muscle protein 22α (SM22α), a contractile protein, and extracellular matrix components elastin and matrix Gla protein (MGP), as well as vascular endothelial growth factor (VEGF). After seeded on the SF-PHBHHx scaffold, the cells showed excellent metabolic activity and proliferation. Conclusion: SPCs isolated from peripheral blood can be differentiated into the SMCs in vitro and have an impressive growth potential in the biodegradable synthesized scaffold. Thus, SPCs may be a promising cell source for constructing TEBVs.

Inhibitory effect of dimeric β peptide on the recurrence and metastasis of hepatocellular carcinoma in vitro and in mice

AIM:To block the adhesion of tumor cells to the extra- cellular matrix, and prevent tumor metastasis and recur- rence, the dimer of the β peptide (DLYYLMDLSYSMKG- GDLYYLMDLSYSMK, β2) was designed and synthesized and its anti-adhesion and anti-invasion effects on hepa- tocellular carcinoma cells were assessed. Additionally, its influence on the metastasis and recurrence of mouse hepatocellular carcinoma was measured. METHODS:The anti-adhesion effect of β2 on the highly metastatic hepatocellular carcinoma cell line HCCLM6 cells and fibronectin (FN) was assayed by the MTT as- say. The inhibition of invasion of HCCLM6 cells by β2 was observed using a Transwell (modified Boyden chamber) and matrigel. Using the hepatocellular carcinoma metas- tasis model and LCI-D20 nude mice, the influence of β2 on the metastasis and recurrence of hepatocellular carci- noma after early resection was investigated. RESULTS:HCCLM6 cells co-incubated with 100 mmol/L, 50 mmol/L, 20 mmol/L or 10 mmol/L β2 for 3 h showed an obvious decrease in adhesion to FN. The adhesion inhibition ratios were 11.8%, 21.7%, 29.6% and 48.7%, respectively. Additionally, HCCLM6 cells cultured with 100 mmol/L β2 had a dramatic decrease in cell invasion. β2 was also observed to inhibit the incisal edge recur- rence and the distant metastasis of nude mice hepato- cellular carcinoma after early resection (P < 0.05). CONCLUSION:The β2 peptide can specifically block the adhesion and invasion of HCCLM6 cells, and can inhibit HCC recurrence and metastasis of LCI-D20 model pos-thepatectomy in vivo. Thus, β2 should be further studied as a new anti-tumor drug.