#407 Genomic and Proteomic Characterization of the Progenitor Population in Cellular Allograft Bone

General Session: What's New in Biologics and Biomechanics

Presented by: T. Moseley

Author(s):

T.A. Moseley (1)
G.M. Williams (1)
E.M. Erbe (1)

(1) NuVasive, Inc., Biologics R&D, San Diego, CA, USA

Abstract

Introduction: The attractiveness of autograft for bone grafting is counterbalanced by donor site morbidity and associated complications(1). Novel grafting materials such as cellular allograft (Osteocel® Plus) are composed of viable cancellous bone in which the cellular component is preserved combined with demineralized bone matrix. This is designed to mimic autograft bone and its properties of osteoconductivity, osteoinductivity and osteogenicity. Previous studies have demonstrated that the cells are a combination of MSCs and osteoprogenitors2. The gene expression of the cells can be utilized to identify how these cells orchestrate osteogenesis. We employed a whole genome array to identify specific genes related to bone formation. The genes were compared to osteoblast and fibroblast RNA. We also demonstrated that these cells can differentiate into osteoblasts using established osteodifferentiation assays.

Methods: Total RNA isolated from six different lots of Osteocel Plus. The mRNA was converted to cRNA which was used to probe microarray BeadChips which contain 24,528 specific human genes according to the manufacturer's instructions. Data analysis included gene identifiers, normalization, transformed signal values for each gene and hypothesis testing using one-way ANOVA or t -Test. Gene expression change of 2.5 fold was determined significant. Additionally, cells enzymatically released from six lots of Osteocel Plus were cultured to 80% confluence in MSC expansion media. The cells were switched to osteodifferentiation media for 10 days and the presence of alkaline phosphatase determined.

Results: Specific genes from Osteocel Plus were expressed differently compared to osteoblasts and fibroblasts. A set of these markers are known to play roles in the differentiation of MSCs toward osteoblasts. The prioritization of these markers, beyond the raw statistical analysis, was performed by referencing known gene expression data3. BMP-2 and BMP-6 were significantly increased over osteoblasts and fibroblasts as was osteocalcin, osteopontin and bone sialoprotein. alkaline phosphatase and crystalline, which are involved in osteoblast differentiation, were increased relative to fibroblasts but not osteoblasts.

All six Osteocel Plus samples were positive for alkaline phosphatase after osteodifferentiation demonstrating that the cells can differentiate into osteoblasts The osteocalcin protein is demonstrated by immunohistochemistry with DAPI nuclear stain.

Discussion: These data indicate that cellular allograft contains a viable progenitor cell population expressing osteogenic genes which are characteristics of MSCs and osteoprogenitors. The BMP genes are known to be osteoinductive and work in synergy with each other. The alkaline phosphatase and crystallin are early markers for osteogenesis and are expressed similarly by Osteocel Plus and osteoblasts. The genes for bone specific matrix proteins, bone sialoprotein, osteocalcin and osteopontin are highly expressed by Osteocel Plus.

Significance: We have contributed evidence that cellular allograft, such as Osteocel Plus, contains cells that express genes related to osteogenesis and are directing bone formation. The demonstration of stem cell allografts compared to autograft will require further studies including their clinical efficacy in well controlled prospective clinical studies.

Table 1: Osteocel Plus Gene Expression