Osteogenic Profiling of Normal Calvarial Bone

For outpatient study recruitment, the research coordinator will review the clinic schedule for potential study candidates. Any patient schedule for a pre-op appointment for surgical calvarial reconstruction will be considered a possible study candidate. The research coordinator will then e-mail the study team the list of potential study candidates with the date and time of the appointment or in-patient study recruitment, the screening procedures include review and collection of information from the patient's medical record-specifically, a physical examination by the plastic surgeon at the Cleft Palate-Craniofacial Center Plastic surgery department. This evaluation will occur either as an inpatient or as an outpatient, depending on the appropriateness and timing of the ultimate surgical repair. During the clinical examination, the plastic surgeon will determine if portions of the skull are unusable for reconstruction. These bone fragments (when too small or shattered to be incorporated into a reconstruction) are normally discarded. It is these fragments alone that will be harvested for evaluation in the present study. The evaluation and surgical repair of these patients requiring post-traumatic reconstruction will not differ from normal, nor from the current standard of care.

Bone samples will be collected from bone tissue that would normally be discarded during the reconstructive surgery, so no additional procedures are needed to collect the bone samples. At the time of surgery one sample of tissue will be taken from bone. The investigator will review the results of the test/procedure that are part of the standard of care and results will become part of the research record. The investigators will continue to use and disclose identifiable medical information and keep tissue samples for seven years. After this seven year period, all identifying information will be removed. Bone marrow tissues will be collected from patients at the time of surgery without any additional dissection or incisions. Bone will be finely minced and then plated in tissue culture flasks as previously reported by other groups.

The procedure will not involve any extra incisions or dissection, as these tissues will be exposed during the reconstructive procedure. At the time of surgery one sample of tissue will be taken from otherwise discarded bone.

Samples will be collected and coded by the plastic surgeon/principal investigator before transfer from the operative room to the Pediatric Craniofacial Biology Laboratory.

The research coordinator will notify Dr. Bykowski of when consented subjects are scheduled for surgery via e-mail. Dr. Bykowski will collect the coded sample and transport them to the Pediatric Craniofacial Biology laboratory.

Cells from each tissue sample will be cultured in vitro. These cells will eventually be kept and frozen in this lab in a temperature locked freezer. The Research Assistant Professor will record subject information into a secure database in Pediatric Craniofacial Biology Laboratory. No information permitting personal identification of family members will be made public. No clinical descriptions that might permit personal identifications will be published, and all clinical information will be stored in secure computer files in the Pediatric Craniofacial Biology Laboratory, which are password protected. Specimens will be stored with assigned code numbers and information linking theses code numbers to the corresponding subjects' identities will also be kept in a password protected database. If a subject should decide to withdraw or be withdrawn from study participation, the linkage code to subject's identity and the specimen will be destroyed.

All cells will be grown in one of two media. Proliferation medium will be used during cell isolation and expansion and will consist of Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin. For mineralization studies, cells will be grown in osteogenic medium (OM) consisting of DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, 10mM β-glycerophosphate, 0.1μM dexamethasone, 50μg/ml ascorbic acid-2-phosphate.

Solid-phase printed arrays of square-shaped 'blots' of immobilized BMP2 will be printed on sialanized fibrin-coated glass cover slips. The blots will be 750 um x 750 um and spaced 1.75 mm apart in a 4 x 4 array. There will be blots with 4 different concentrations of BMP2, ranging from zero to binding site saturation, along each column, and there will be 4 replicates of each column.

The homogenous fibrin films cross-linked to glass slides will be prepared. Blot concentrations will be modulated using the overprinting strategy. Pattern accuracy and surface concentration of growth factors and their persistence will be verified for FGF2. BMP2 bio-inks will be diluted to 10 µg/ml in 20 mM sodium acetate, pH 7.4. Preliminary experiments on immobilization and persistence of BMP2 on fibrin films indicate at least 6 day persistence.

Bio-ink Formulations for BMP2 and Inhibitors. The purpose here is to determine the concentration and to provide initial validation of patterned inhibition response. FGF2 bio-inks will be prepared. All bio-inks will be diluted to a 10 µg/ml. Since the number of binding sites on a square is fixed, the bio-ink dilutions and/or number of overprints may be modified based on initial results to accommodate multiple growth factors now occupying a given square. From the above array studies the investigators will determine the concentrations of BMP2 and inhibitor necessary to show stimulation/inhibition.

Cells will be placed on the printed patterns described above. Cells grown on discreet arrays will be fixed and stained for ALP activity (Sigma) after 1, 3, or 7 days in culture on the growth factor patterns. Mineralization will be tested after culturing cells on patterns in OM for 7, 14, or 21 days using alizarin red staining (for calcium) and von Kossa staining (for phosphates). ALP staining will be performed to identify early osteogenic differentiation. To signify "end" of BMP2 signaling, matrix mineralization will be determined by positive von Kossa or alizarin red staining using established protocols.

BMP2 and potential inhibitor proteins can be deposited simultaneously. The image pattern for jetting the array will be the same as in Figures 1 and 3, with the image pattern for the inhibitor being the same as the BMP2 pattern, only rotated by 90°. The jetting will be coordinated with the stage motions so that these two patterns will exactly overlap using computer-vision calibration. Pattern accuracies will be verified using fluorescently-labeled BMP2 and inhibitors.

The concentrations of BMP2 that are capable of inducing ALP activity and matrix mineralization and the concentrations of inhibitors that are capable of counteracting BMP2-induced osteogenesis will be identified and recorded for each patient. In comparing the respective osteogenic potentials, the investigators will develop "osteogenic profiles" for each of the tissue samples isolated from the patients