ABSTRACT
Objective To investigate the influence of dynamic mechanical stimulation on the annulus fibrosus (AF) cells seeded on silk scaffolds.Methods AF cells were isolated from rabbits and were seeded on the scaffold,then cultured for 3,7,14 days with different range of dynamic compression.Stereomicroscope and scanning electron microscope (SEM) was used to observe the surface morphology of tissue engineering annulus fibrosus cells (TE-AFs).After fixation,samples were harvested for histological staining.AF cells related extracellular matrix (ECM) was evaluated by the quantitative analysis of total DNA,proteoglycan and collagen I.The mechanical properties were compared within different groups.Results Stereomicroscope and SEM results showed that the colors of TE-AFs in all groups were deepening with time going.SEM showed cell adhesion on the scaffold and the secretion of extracellular matrix.Histological,immunohistochemical staining,biochemical quantitative analysis and total DNA content showed that the AF cells inside scaffolds could support AF cell attachment,proliferation and secretion.As a result,the compressive properties were enhanced with increasing culture time.Stereomicroscope showed that the colors of TE-AFs in all groups were deepening with time going after dynamic compression.HE staining,Safranin O staining and Type Ⅰ collagen staining showed that cell proliferation and secretion,GAG secretion and collagen secretion were increased with time going within different groups.Quantitation of GAG achieved maximum in 15％ strain group,and quantitation of collagen achieved maximum in 10％ strain group.The total DNA content achieved maximum in 5％ strain group,and compression elastic modulus achieved maximum in 15％strain goup.The height of TE-AFs did not change after mechanical stimulation for 14 days.Conclusion Suitable mechanical stimulation is a positive factor for new AF tissue engineering that will tend to the nature tissue.Excessive compression can accelerate the progress of cell apoptosis.
ABSTRACT
Objective The biomimetic osteochondral scaffold contained calcified cartilage layer(CCL) was fabricated using slik fibroin (SF) and hydroxyapatite (HA) for materials.To investigate effects of biomimetic osteochondral scaffold contained CCL compounding with ADSCs on regeneration of the osteochondral defect on the rabbit knee,explore the feasibility of this design as a concept of osteochondral tissue engineering.Methods We fabricated a novel biomimetic osteochondral scaffold with CCL using SF and HA by the combination of paraffin-sphere leaching and modified temperature gradient-guided thermal-induced phase separation (TIPS) technique.The pore size,porosity,and compressive modulus of elasticity of the scaffold cartilage layer and the osteogenic layer were measured by scanning electron microscopy and microscopy CT.The osteochondral defect model on rabbit bilateral knees were established,and implanted with the non-CCL group (non-CCL scaffold compounding with ADSCs) and CCL group (CCL scaffold compounding with ADSCs).At 4,8 and 12 weeks after implantation,the rabbits were euthanized,respectively.Gross observation score,histological and immunohistochemical assessment,biochemical quantitative of new osteochondral tissue,micro-CT scans for new bone,were executed.We evaluated the regeneration of osteochondral defects in each group,and verified the role of CCL in vivo.Results The biomimetic osteochondral scaffold with CCL had a consecutively overlapping trilayer structure with different densities and pore structures,including a chondral layer (top layer),intermediate layer and bony layer (bottom layer).The cartilage layer had a well-oriented microporous structure with a uniform distribution with a pore size of (112.43± 12.65)μm and a porosity of 90.25％±2.05％.The subchondral bone layer had a good three-dimensional macroporous structure,good connectivity,pore size (362.23±26.52) μm,porosity of osteogenic layer was 85.30％± 1.80％.The cartilage regeneration in CCL+AD-SCs group was better than non-CCL+ADSCs group.The content of GAG and type Ⅱ collagen in new cartilage tissue in CCL+AD-SCs group was much more than non-CCL+ADSCs group.The new bone tissue analysis and biomechanical testing had no significant differences between the two groups.Conclusion The biomimetic osteochondral SF/HA scaffold contained CCL mimics the structure of normal osteochondral tissue with good 3-dimensional pore structure and biocompatibility.The scaffold complex autologous ADSCs successfully repair osteochondral defects in rabbit knee,and the presence of CCL accelerates the growth of cartilage.