Sunday, October 10, 2004

A Gene Expression Analysis of Human Mandibular Distraction Callus

Rian A Maercks, MD, XP Reyna Rodriguez, DDS, Jennifer Marler, MD, Cathy Ebert, MS, LE Landa, MD, DMD, Enrique Ochoa Lopez Diaz, MD, and Christopher B Gordon, MD.

Purpose:  Rapid distraction in the craniofacial skeleton produces bony callus at rates that are unsurpassed in any other system.  This provides a unique opportunity to identify novel gene expression patterns in human mandibular distraction callus, identify families of genes that are co-regulated, and may help identify target genes for therapeutic intervention. Materials/Methods: Seven patients with micrognathia underwent mandibular distraction using the “No Vector” technique (Ages 1-20 months).  Samples of normal bone and adjacent distraction callus were harvested at time of initial osteotomy and at removal of the distractor.  These samples were protected with RNA Later (Qiagen), total RNAs were isolated and Code Link (Amersham) labeled cRNAs were generated for array hybridization.  The Affymetrix U133 human genome array was used to analyze over 47,000 transcripts representing approximately 33,000 genes.  Data analysis was completed with GeneSpring software ( Silicone Genetics).  The data were then normalized using multiple internal controls including serial time points, and levels of other ubiquitously expressed genes. Experience:  We have completed data collection and analysis of seven patients who underwent successful mandibular distraction for retrognathia.  Each patient had successful distraction with a mean distraction distance of 41mm.  Discarded tissues from the time of initial osteotomy and at the time of subsequent callus manipulation were harvested and directly prepared for RNA analysis.  The later time points corresponded to active distraction phase and consolidation phase. Results:  We have identified 407 genes that consistently demonstrated a ten-fold or greater change in expression in the distracted tissue compared with the nascent tissue.  We confirm a correlation with current literature citing TGF beta and related factors, ECM proteins and BMPs as essential mediators of distraction.  Of this group, there were also 50 expressed sequence tags that represent ostensibly unique new genes.  There were several new genes which by predicted structure represent novel collagen isoforms or other unique matrix molecules.  Surprisingly collagen 1 expression was not significantly altered during distraction.  Collagen III, IV and VI are among the most familiar genes in the overexpressed group.  We also find robust expression of several BMPs other than the BMP-2,4,7 pattern previously identified in animal models.  Table one highlights eleven genes previously identified in the literature as important in the distraction process.  

Table 1 Relative expression of genes previously implicated in distraction osteogenisis Gene                                       fold change

TGF-beta                               >10X up

Osteonectin                          3 X up

Timp1                                     3 X up

IGF 2                                       2 X up

BMP 4                                    1 X up

Col A1                                    no change

FGF 2                                      no change

BMP 2                                    1 X down

BMP 7                                    2 X down

Osteopontin                          2 X down

Osteocalcin                           4 X down                                                              


Conclusions: In this study we use microarray technology to screen human mandibular distraction callus for gene expression alterations.  We have observed changes in familiar cytokines that are consistent with current literature.  We have also identified many novel genes and several unexpected gene expression patterns.  Early results suggest that dedifferentiation in the bony callus may represent a way to harness potential bony precursor cells.  Current studies in our laboratory will characterize promising candidate genes to develop new therapeutic strategies in bone healing and tissue engineering.