ABSTRACT
BACKGROUND: Anterior cruciate ligament (ACL) injury is commonly seen, and mainly treated with ACL reconstruction. Ligament tissue engineering treatment for ACL injury has become the current focus due to the extensive use of tissue engineering techniques in clinical practice. OBJECTIVE: To investigate the biocompatibility of human amniotic mesenchymal stem cells (hAMSCs) and human acellular amniotic membrane(HAAM)scaffolds in vitro. METHODS: hAMSCs were isolated using enzyme digestion and then identified. The fresh human amniotic membrane was subjected to decellularization using enzymatic digestion and chemical decontamination. Hematoxylin-eosin staining and immunofluorescence were used to detect the removal of cell components and destruction of the extracellular matrix. hAMSCs were cultured with HAAM extract (experimental group) and normal L-DMEM/F12 medium (control group). Cell proliferation was detected by cell counting kit-8. Cell adhesion and growth were observed by scanning electron microscope and inverted microscope at 14 days after co-culture with HAAM. RESULTS AND CONCLUSION: hAMSCs exhibited a spiral, adherent growth under the inverted phase contrast microscope; and the cells highly expressed vimentin and lowly expressed CK-19, with osteogenic, adipogenic, and chondrogenic potential. Hematoxylin-eosin staining showed that human acellular amniotic epithelial cells and mesenchymal stem cells were completely removed and the extracellular matrix had no obvious damage. Immunofluorescence staining showed positive expression of type I collagen and type III collagen. The HAAM extract had no cytotoxicity to hAMSCs and exerted no effect on its proliferative activity. HAAM could promote the proliferation and adhesion of hAMSCs after incubation under the inverted microscope and scanning electron microscope. These findings indicate that HAAM has good biocompatibility and is favorable for the proliferation of hAMSCs.
ABSTRACT
BACKGROUND: Tendon-to-bone healing is a complex and slow process, which often hinders the therapeutic outcomes. To enhance the tendon-to-bone healing, increasing cell bioactivity on the contact surface is a new strategy. Hypoxia-inducible factor 1α (HIF-1α) can induce the formation of blood vessel and bone tissue via many ways.However,it is unclear whether HIF-1α can strengthen differentiation of human amniotic mesenchymal stem cells (hAMSCs) induced by Scleraxis, a specific marker of tendon, and can be used in tendon and bone repair. OBJECTIVE: To investigate the ability of HIF-1α in enhancing Scleraxis to modify hAMSCs aiming to promote tendon-to-bone healing and its molecular mechanism. METHODS: Passage 3 hAMSCs were infected with AdHIF-1α, AdScx, AdGFP and AdHIF-1α+AdScx. The expressions of related genes of tendon, cartilage and bone tissue were detected at 3 and 7 days after infection. RESULTS AND CONCLUSION: Under the inverted phase contrast microscopy, the passage 3 hAMSCs were in spindle shape and presented with vortex-like adherent growth. Under the transmission electron microscopy, hAMSCs were in oval shape and had clear structure, with abundant endoplasmic reticulum and mitochondria. Fluorescence photographs were taken at 24 hours after adenovirus infection, showing about 50% red fluorescence expression in the AdHIF-1α group, while about 70% green fluorescence expression in the AdScx and AdGFP groups. Real time-PCR results showed that at 3 and 7 days after infection, the mRNA expressions of collagen type I, Fibronectin, RUNX2, VEGF and ALP in the AdHIF-1α+AdScx group, AdHIF-1α group and AdScx group were higher than those in the AdGFP group (P < 0.05); the mRNA expressions of collagen type I, Fibronectin, RUNX2, VEGF and ALP in the AdHIF-1α+AdScx group were significantly higher than those in the AdScx group (P < 0.05). Fluorescence immunohistochemistry results showed that the expression of collagen type I in the AdHIF-1α+AdScx group at 7 days after infection was higher than that at 3 days after infection. To conclude, HIF-1α can enhance the ability of Scleraxis to modify hAMSCs to promote tendon-to-bone healing by upregulating the expressions of molecular markers of tenocytes, chondrocytes and osteocytes.