Objective To fabricate a novel composite scaffold with acellular demineralized bone matrix/acellular nucleus pulposus matrix and to verify the feasibility of using it as a scaffold for intervertebral disc tissue engineering through detecting physical and chemical properties. Methods Pig proximal femoral cancellous bone rings (10 mm in external diameter, 5 mm in internal diameter, and 3 mm in thickness) were fabricated, and were dealed with degreasing, decalcification, and decellularization to prepare the annulus fibrosus phase of scaffold. Nucleus pulposus was taken from pig tails, decellularized with Triton X-100 and deoxycholic acid, crushed and centrifugalized to prepare nucleus pulposus extracellular mtrtix which was injected into the center of annulus fibrosus phase. Then the composite scaffold was freeze-dryed, cross-linked with ultraviolet radiation/carbodiimide and disinfected for use. The scaffold was investigated by general observation, HE staining, and scanning electron microscopy, as well as porosity measurement, water absorption rate, and compressive elastic modulus. Adipose-derived stem cells (ADSCs) were cultured with different concentrations of scaffold extract (25%, 50%, and 100%) to assess cytotoxicity of the scaffold. The cell viability of ADSCs seeded on the scaffold was detected by Live/Dead staining. Results The scaffold was white by general observation. The HE staining revealed that there was no cell fragments on the scaffold, and the dye homogeneously distributed. The scanning electron microscopy showed that the pore of the annulus fibrosus phase interconnected and the pore size was uniform; acellular nucleus pulposus matrix microfilament interconnected forming a uniform network structure, and the junction of the scaffold was closely connected. The novel porous scaffold had a good pore interconnectivity with (343.00 ± 88.25) µm pore diameter of the annulus fibrosus phase, 82.98% ± 7.02% porosity and 621.53% ± 53.31% water absorption rate. The biomechanical test showed that the compressive modulus of elasticity was (89.07 ± 8.73) kPa. The MTT test indicated that scaffold extract had no influence on cell proliferation. Live/Dead staining showed that ADSCs had a good proliferation on the scaffold and there was no dead cell. Conclusion Novel composite scaffold made of acellular demineralized bone matrix/acellular nucleus pulposus matrix has good pore diameter and porosity, biomechanical properties close to natural intervertebral disc, non-toxicity, and good biocompatibility, so it is a suitable scaffold for intervertebral disc tissue engineering.
Objective To explore the preparing methods in vitro and test the cl inical appl icabil ity of implantation in vivo of bone marrow stromal stem cells (BMSCs)-biphasic scaffold to repair defects of cartilage and subchondral bone and tocompare the differences in repaired outcomes of composite, single biphasic scaffold and rabbits themselves. Methods The upper chondral phase and the lower osseous phase of the plugs, using poly-lactic-co-glycol ic acid (PLGA), hydroxyapatite (HA), and other biomaterials, were fused into carrier scaffold, on which collagen type I (Col I) was coated. The surface and inner structure of bi phasic scaffold were observed under scanning electron microscope (SEM). BMSCs was isolated from the bone marrow of tibia and femurs of young New Zealand rabbits using centrifuging and washing, and their morphologies and adherences were observed everyday. Then BMSCs were inoculated on the surface of scaffold to form BMSCs-scaffold composites. Osteochondral defects were surgically created on articular surface of femoral intercondyles of 30 New Zealand rabbits, which were divided into groups A, B and C. In group A, a bi phasic osteochondral composite were implanted into defect, BMSCs and biphasic cyl indrical porous plug of PLGA-HA-Col I in group B, and group C was used as a control without implant. Specimens were harvested to make macroscopic and histological observations at the 1st, 3rd, 6th, and 9th months after operation respectively; meanwhile immunohistological and micro-computed tomography (micro CT) examinations were performed and graded at the 9th month after operation. Results SEM showed an excellent connection of holes in the biphasic scaffold infiltrated by Col I. Optical microscopy and SEM showed a good growth of BMSCs in scaffold without obvious cellular morphological changes and an accumulation in the holes. Macroscopic samples showed a resistant existence of defects of group C within 9 months; the scaffold completely degenerated and chondral-l ike tissue formed on articular surface with partly collapses and irregular defects in group A; and smoother surface without collapses and approach to normal with texture of new regeneration in group B. There were statistically significant differences in macroscopic results (P lt; 0.001), group B was superior to group A, and group C was the worst. The micro CT showed good repairs and reconstruction of subchondral bone, with a acceptable integration with newborn chondral-l ike tissue and host bone in group B. Quantificational analysis of relevantparameters showed no significant differences. Histological results showed inflammations located in defects at the 1st month, new tissue grew into scaffold at the 3rd month; new chondral-l ike tissue crept on the margin of defects and biphasic scaffold degenerated completely at the 6th month, and lots of collagen formed in subchondral bone with major fibrocartilage on chondralarea at the 9th month after surgery in groups A and B. In groups A and B, immunohistological observations were weak positive for Col II and positive for Col I. Conclusion Biphasic scaffold implanted in body can induce and accelerate repair of defects of articular cartilages which are mainly filled with fibrocartilage, especially for subchondral bone. Scaffold combined with BMSCs has the best repairing effects 9 months after implantation.