Effect of bone graft substitute on marrow stromal cell proliferation and differentiation.

Marrow stromal cells (MSCs) are ideally suited for tissue engineered bone grafts since they have the potential to regenerate bone, but may also maintain the homeostasis of the repaired tissue through their ability for self-renewal. An ideal bone graft substitute should support MSC self-renewal as well as differentiation to ensure complete bone defect regeneration and maintenance. The purpose of this investigation was to determine the effect of different substrate materials on MSC expansion and differentiation. Calcium polyphosphate (CPP), bone and hydroxyapatite/tricalcium phosphate (HA/TCP) were seeded with rat MSCs and maintained in culture conditions that promote cell expansion. At 0, 3, 7, 14, and 21 days cell numbers were determined by measuring their metabolic activity using a MTT assay and the frequency of cycling cells by 24 hr BrdU incorporation. Osteogenic, chondrogenic, and adipogenic marker expression in these cultures was measured by qRT-PCR. An initial drop in cell numbers was observed on all substrates. CPP and bone, but not HA/TCP supported an increase in proliferating cells at day 14 and 21. In addition, no upregulation of mature bone markers was observed in cells cultured on CPP and bone, which suggests that these substrates support the expansion of undifferentiated MSCs. In contrast, cell numbers on HA/TCP decreased with time and only rare BrdU positive cells were observed. This decrease in proliferation correlated with the down regulation of osteogenic progenitor markers and the substantial increase in mature osteocyte markers, indicating that HA/TCP favors MSC differentiation and maturation along the osteogenic lineage.

A repeatable ex vivo model of spondylolysis and spondylolisthesis.

STUDY DESIGN: An ex vivo biomechanical study using porcine spinal segments. OBJECTIVE: To produce a biomechanical model of both spondylolysis and spondylolisthesis using an accelerated cyclic loading model with intermittent impulse loads. SUMMARY OF BACKGROUND DATA: Only a few models of spondylolisthesis appropriate for biomechanical testing have been presented previously. Past modeling attempts have largely required nonphysiologic gross fracture of the pars before testing and have resulted in nonphysiologic endplate fracture. In these tests no clinically relevant spondylolisthesis was seen at the end of testing. A reproducible, clinically relevant model of both spondylolysis and spondylolisthesis would allow study of these disease processes, and facilitate the development and evaluation of advanced spinal implants optimized specifically for these pathologies. METHODS: Five porcine lumbar functional spinal units were tested (2 L4-L5, 3 L6-S1) after small notches had been created in the pars and after the disc had specific collagen fibers in the anterior anulus sectioned. Specimens were loaded with a constant cranial-caudal compressive force of 300 N and the application of cyclic anterior shear loads between 300 and 600 N with intermittent impulse loads to 1500 N until pars fracture occurred. Elevated cyclic loading then continued between 500 and 800 N. RESULTS: All specimens displayed bilateral pars fracture with the fractures passing through the points of notching and no damage to endplates or facet joints. Clinically-relevant Grade II spondylolisthesis was achieved in all 5 specimens. The mean slip at the conclusion of testing was 33%. CONCLUSION: Cyclic shear loading with intermittent impulse loads can reliably create fracture in the pars interarticularis in ex vivo porcine spine segments. Subsequent cyclic anterior motion of the superior vertebra results in clinically-relevant spondylolysis and spondylolisthesis.

Preparation of BMP-2 containing bovine serum albumin (BSA) nanoparticles stabilized by polymer coating.

PURPOSE: The purpose of this study was to investigate the preparation process of bone morphogenetic protein-2 (BMP-2) containing bovine serum albumin (BSA) nanoparticles (NPs), and to assess the bioactivity of BMP-2 encapsulated in such NPs. METHODS: The NPs were prepared by a coacervation method, and the effects of process parameters on NP size and polydispersity were examined. Polymer coated NPs were characterized with respect to amount of adsorbed polymer, particle size and zeta potential. Using bone marrow stromal cells (BMSC), biocompatibility of the NPs was investigated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) Assay, and bioactivity of the encapsulated BMP-2 was investigated by alkaline phosphatase (ALP) induction and calcification. RESULTS: The size of NPs could be controlled in the 50-400 nm range by process parameters including BSA concentration, non-solvent:solvent ratio and pH value. After coating with cationic polymers, the particle size and zeta potential were significantly increased. MTT assay indicated no toxicity of both the uncoated and coated NPs on BMSC. Based on ALP induction and calcification, full retention of BMP-2 bioactivity was retained in the polymer-coated NPs. CONCLUSIONS: This study described a preparation procedure for BSA NPs with controllable particle size, and such polymer-coated BSA NPs are promising delivery agents for local and systemic administration of BMP-2 in bone regeneration.