Abstract
Craniosynostosis is a condition caused by premature fusion of one or more cranial sutures during fetal development. Inappropriate suture fusion can lead to abnormal skull shape and increased intracranial pressure, as well as impaired circulation, breathing, vision, hearing, swallowing, and cognition. The severity of craniosynostosis varies according to which sutures are affected, and how early the fusion events occur. Craniosynostosis can occur as a part of a syndrome or as an isolated, nonsyndromic condition. Nonsyndromic craniosynostosis accounts for approximately 80% of craniosynostosis cases, though the etiologic factors remain somewhat elusive. Single nucleotide polymorphism (SNP) array data has found a SNP in the promoter region of Bone Morphogenetic Protein 2 (BMP2) in nonsyndromic sagittal craniosynostosis. It is unclear how the SNP causes BMP2 to be transcriptionally upregulated, but one hypothesis is that increased BMP2 induces embryonic mesenchymal cells to differentiate into osteoblasts early to promote premature suture fusion. Here, I describe my research centered on investigating the connection between increased BMP2 and early differentiation of mesenchymal precursor cells into osteoblasts. Cells found in the sutures of infants with nonsyndromic single suture craniosynostosis were characterized to determine if they were mesenchymal stem cells (MSCs). I also tested the hypothesis that cells from open sutures of nonsyndromic single suture craniosynostosis patients are more closely related to wild type (WT) bone marrow MSCs, while the cell population from the prematurely fused sutures are more differentiated toward the osteoblast lineage due to altered BMP2 transcription. Although the critical SNP within the BMP2 promoter region was present in all nonsyndromic craniosynostosis patient samples tested, we hypothesized that differences in paracrine factors can influence the expression pattern of BMP2 in conjunction with the promoter region SNP. Samples of human skull fragments from nonsyndromic single suture craniosynostosis patients were obtained from surgeries performed at the UC Davis Medical Center. Cells were extracted from the bone storage media, from the protease digestion process, and directly from skull bone fragments. Analysis using flow cytometry indicated that all of the extracted cells were MSCs. MTT assays, cell counts, and CFU-F assays were used to determine cell proliferation differences between the open and fused suture MSCs. These experiments suggested that fused suture MSCs had higher rates of proliferation than open suture MSCs, which underwent cell division at a rate similar to WT MSCs. To determine the osteogenic differentiation potential of the suture cells, I performed Alizarin Red S assays, which indicated that fused suture cells produced more calcium deposits. Alkaline phosphatase (ALP) activity assays were used to further investigate the ability of the MSCs to differentiate into bone. These assays showed that open suture cells have higher ALP activity in comparison to both open suture cells and WT MSCs. Lastly, qPCR was conducted to examine gene expression differences between open and fused suture cells. It was shown that fused suture cells had decreased expression of BSP, osteocalcin, and osteopontin and increased expression of ALP. These findings indicate that the differences observed between fused and open suture cells could be due to multiple causes. The fused suture cells could be prematurely specified toward the osteoblast lineage, and could therefore be differentiating into osteoblasts at a much earlier time than open suture cells. Additionally, the fused suture cells could be dysfunctional in terms of cell division regulation. Due to the many variables associated with using human patient samples, more research should be performed in order to elucidate the complex causation of nonsyndromic single suture craniosynostosis.