Publications by authors named "Mark P Mooney"

92 Publications

Interleukin-10 Does Not Augment Osseous Regeneration in the Scarred Calvarial Defect Achieved with Low-Dose Biopatterned BMP2.

Plast Reconstr Surg 2019 06;143(6):1215e-1223e

From the Department of Plastic Surgery, University of Pittsburgh; Biomedical Engineering and Biological Sciences and The Robotics Institute, Carnegie Mellon University; and The Ohio State University College of Medicine.

Background: Large calvarial defects represent a major reconstructive challenge, as they do not heal spontaneously. Infection causes inflammation and scarring, further reducing the healing capacity of the calvaria. Bone morphogenetic protein-2 (BMP2) has been shown to stimulate osteogenesis but has significant side effects in high doses. BMP2 has not been tested in combination with antiinflammatory cytokines such as interleukin-10.

Methods: Sixteen New Zealand White rabbits underwent 15 × 15-mm flap calvarectomies. The flap was incubated in Staphylococcus aureus and replaced, and infection and scarring were allowed to develop. The flap was subsequently removed and the wound débrided. A 15 × 15-mm square of acellular dermal matrix biopatterned with low-dose BMP2, interleukin-10, or a combination was implanted. Computed tomographic scans were taken over 42 days. Rabbits were then killed and histology was performed.

Results: Defects treated with BMP2 showed significantly (p < 0.05) greater osseous regeneration than untreated controls. Interleukin-10 did not significantly augment the healing achieved with BMP2, and interleukin-10 alone did not significantly increase healing compared with controls. Histology showed evidence of bone formation in defects treated with BMP2. Untreated controls and defects treated with interleukin-10 alone showed only fibrous tissue in the defect site.

Conclusions: Low-dose BMP2 delivered directly to the scarred calvarial defect augments bony healing. Interleukin-10 at the dose applied did not significantly augment healing alone or in combination with BMP2. Healing had not finished at 42 days and analysis at later time points or the use of higher doses of BMP2 may yield greater healing.
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http://dx.doi.org/10.1097/PRS.0000000000005640DOI Listing
June 2019

Molecular Analyses in a Rabbit Model of Craniosynostosis: Likely Exclusion of Known Candidate Genes as the Loci of Origin.

Cleft Palate Craniofac J 2019 07 28;56(6):786-790. Epub 2018 Oct 28.

1 Department of Plastic Surgery, University of Pittsburgh/Children's Hospital of Pittsburgh, Pittsburgh, PA, USA.

Objective: Craniosynostosis (CS) involves the premature fusion of one or more cranial sutures. We work with a naturally occurring rabbit model of CS with an undefined etiology. Known causes of coronal CS were evaluated to identify potential associations with CS in the rabbit.

Design: Candidate genes were sequenced in control New Zealand White (NZW) rabbits (n = 4) and synostotic NZW rabbits (n = 4). Variants were identified by alignment using Clustal Omega.

Outcome Measures: Single nucleotide variants (SNVs) were classified according to phenotypic associations and predicted impact on protein structure. Human correlates were identified in the database of single nucleotide polymorphisms (dbSNP).

Results: A total of 21 SNVs were identified in the 10 genes examined. Variant classification and inheritance patterns are inconsistent with causality.

Conclusions: The genetic basis for disease in the CS rabbit likely involves novel loci and is not associated with known causes of coronal synostosis.
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http://dx.doi.org/10.1177/1055665618808623DOI Listing
July 2019

Reconstruction of a Calvarial Wound Complicated by Infection: Comparing the Effects of Biopatterned Bone Morphogenetic Protein 2 and Vascular Endothelial Growth Factor.

J Craniofac Surg 2019 Jan;30(1):260-264

Department of Plastic Surgery, University of Pittsburgh.

Bone morphogenetic protein 2 (BMP2) bioprinted on biological matrix induces osseous regeneration in large calvarial defects in rabbits, both uncomplicated and scarred. Healing in unfavorable defects scarred from previous infection is decreased due in part to the lack of vascularity. This impedes the access of mesenchymal stem cells, key to osseous regeneration and the efficacy of BMP2, to the wound bed. The authors hypothesized that bioprinted vascular endothelial growth factor (VEGF) would augment the osseous regeneration achieved with low dose biopatterned BMP2 alone. Thirteen New Zealand white rabbits underwent subtotal calvariectomy using a dental cutting burr. Care was taken to preserve the underlying dura. A 15 mm × 15 mm flap of bone was cut away and incubated in a 1 × 108 cfu/mL planktonic solution of S aureus before reimplantation. After 2 weeks of subsequent infection the flap was removed and the surgical wound debrided followed by 10 days of antibiotic treatment. On postoperative day 42 the calvarial defects were treated with acellular dermal matrix bioprinted with nothing (control), VEGF, BMP2, BMP2/VEGF combined. Bone growth was analyzed with serial CT and postmortem histology. Defects treated with BMP2 (BMP2 alone and BMP2/VEGF combination) showed significantly greater healing than control and VEGF treated defect (P < 0.5). Vascular endothelial growth factor treated defect demonstrated less healing than control and VEGF/BMP2 combination treatments achieved less healing than BMP2 alone though these differences were nonsignificant. Low dose BMP2-patterned acellular dermal matrix improves healing of scarred calvarial defects. Vascular endothelial growth factor at the doses applied in this study failed to increase healing.
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http://dx.doi.org/10.1097/SCS.0000000000004779DOI Listing
January 2019

Genetic associations and phenotypic heterogeneity in the craniosynostotic rabbit.

PLoS One 2018 20;13(9):e0204086. Epub 2018 Sep 20.

Department of Plastic Surgery, University of Pittsburgh/Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

Craniosynostosis (CS) is a disorder that involves the premature ossification of one or more cranial sutures. Our research team has described a naturally occurring rabbit model of CS with a variable phenotype and unknown etiology. Restriction-site associated DNA (RAD) sequencing is a genomic sampling method for identifying genetic variants in species with little or no existing sequence data. RAD sequencing data was analyzed using a mixed linear model to identify single nucleotide polymorphisms (SNPs) associated with disease occurrence and onset in the rabbit model of CS. SNPs achieving a genome-wide significance of p ≤ 5 x 10-8 were identified on chromosome 2 in association with disease occurrence and on chromosomes 14 and 19 in association with disease onset. Genotyping identified a coding variant in fibroblast growth factor binding protein 1 (FGFBP-1) on chromosome 2 and a non-coding variant upstream of integrin alpha 3 (ITGA3) on chromosome 19 that associated with disease occurrence and onset, respectively. Retrospective analysis of patient data revealed a significant inverse correlation between FGFBP-1 and ITGA3 transcript levels in patients with coronal CS. FGFBP-1 and ITGA3 are genes with roles in early development that warrant functional study to further understand suture biology.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0204086PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6147457PMC
March 2019

The influence of suturectomy on age-related changes in cerebral blood flow in rabbits with familial bicoronal suture craniosynostosis: A quantitative analysis.

PLoS One 2018 1;13(6):e0197296. Epub 2018 Jun 1.

Departments of Oral Biology, University of Pittsburgh, Pittsburgh, PA, United States of America.

Background: Coronal suture synostosis is a condition which can have deleterious physical and cognitive sequelae in humans if not corrected. A well-established animal model has previously demonstrated disruptions in intracranial pressure and developmental abnormalities in rabbits with congenital craniosynostosis compared to wild type rabbits.

Objective: The current study aimed to measure the cerebral blood flow (CBF) in developing rabbits with craniosynostosis who underwent suturectomy compared to those with no intervention and compared to wild type rabbits.

Methods: Rabbits with early onset coronal suture synostosis were assigned to have suturectomy at 10 days of age (EOCS-SU, n = 15) or no intervention (EOCS, n = 18). A subset of each group was randomly selected for measurement at 10 days of age, 25 days of age, and 42 days of age. Wild type rabbits (WT, n = 18) were also randomly assigned to measurement at each time point as controls. Cerebral blood flow at the bilateral hemispheres, cortices, thalami, and superficial cortices was measured in each group using arterial spin-labeling MRI.

Results: At 25 days of age, CBF at the superficial cortex was significantly higher in EOCS rabbits (192.6 ± 10.1 mL/100 mg/min on the left and 195 ± 9.5 mL/100 mg/min on the right) compared to WT rabbits (99.2 ± 29.1 mL/100 mg/min on the left and 96.2 ± 21.4 mL/100 mg/min on the right), but there was no significant difference in CBF between EOCS-SU (97.6 ± 11.3 mL/100 mg/min on the left and 99 ± 7.4 mL/100 mg/min on the right) and WT rabbits. By 42 days of age the CBF in EOCS rabbits was not significantly different than that of WT rabbits.

Conclusion: Suturectomy eliminated the abnormally increased CBF at the superficial cortex seen in EOCS rabbits at 25 days of age. This finding contributes to the evidence that suturectomy limits abnormalities of ICP and CBF associated with craniosynostosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197296PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983410PMC
November 2018

Maternal environment and craniofacial growth: geometric morphometric analysis of mandibular shape changes with in utero thyroxine overexposure in mice.

J Anat 2018 07 2;233(1):46-54. Epub 2018 Apr 2.

Department of Oral Health Sciences, Medical University of South Carolina, Charleston, South Carolina, USA.

An estimated 3% of US pregnancies are affected by maternal thyroid dysfunction, with between one and three of every 1000 pregnancies being complicated by overactive maternal thyroid levels. Excess thyroid hormones are linked to neurological impairment and excessive craniofacial variation, affecting both endochondral and intramembranous bone. Using a geometric morphometric approach, this study evaluates the role of in utero thyroxine overexposure on the growth of offspring mandibles in a sample of 241 mice. Canonical variate analysis utilized 16 unilateral mandibular landmarks obtained from 3D micro-computed tomography to assess shape changes between unexposed controls (n = 63) and exposed mice (n = 178). By evaluating shape changes in the mandible among three age groups (15, 20 and 25 days postnatal) and different dosage levels (low, medium and high), this study found that excess maternal thyroxine alters offspring mandibular shape in both age- and dosage-dependent manners. Group differences in overall shape were significant (P < 0.001), and showed major changes in regions of the mandible associated with muscle attachment (coronoid process, gonial angle) and regions of growth largely governed by articulation with the cranial base (condyle) and occlusion (alveolus). These results compliment recent studies demonstrating that maternal thyroxine levels can alter the cranial base and cranial vault of offspring, contributing to a better understanding of both normal and abnormal mandibular development, as well as the medical implications of craniofacial growth and development.
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http://dx.doi.org/10.1111/joa.12810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5987819PMC
July 2018

Molecular Analysis of Gli3, Ihh, Rab23, and Jag1 in a Rabbit Model of Craniosynostosis: Likely Exclusion as the Loci of Origin.

Cleft Palate Craniofac J 2018 03 18;55(3):375-382. Epub 2017 Dec 18.

1 Department of Plastic Surgery, Children's Hospital of Pittsburgh, University of Pittsburgh, Pittsburgh, PA, USA.

Objective: Craniosynostosis (CS) involves the premature fusion of one or more cranial sutures. The etiology of CS is complex and mutations in more than 50 distinct genes have been causally linked to the disorder. Many of the genes that have been associated with CS in humans play an essential role in tissue patterning and early craniofacial development. Among these genes are members of the Hedgehog (HH) and Notch signal transduction pathways, including the GLI family member Gli3, Indian Hedgehog ( Ihh), the RAS oncogene family member Rab23, and the Notch ligand JAGGED1 ( Jag1). We have previously described a colony of rabbits with a heritable pattern of coronal suture synostosis, although the genetic basis for synostosis within this model remains unknown. The present study was performed to determine if coding errors in Gli3, Ihh, Rab23, or Jag1 could be causally linked to craniosynostosis in this unique animal model.

Design: Sequencing of cDNA templates was performed using samples obtained from wild-type and craniosynostotic rabbits.

Results: Several nucleotide polymorphisms were identified in Gli3, Ihh, and Rab23, although these variants failed to segregate by phenotype. No nucleotide polymorphisms were identified in Jag1.

Conclusions: These data indicate that the causal locus for heritable craniosynostosis in this rabbit model is not located within the protein coding regions of Gli3, Ihh, Rab23, or Jag1.
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http://dx.doi.org/10.1177/1055665617739001DOI Listing
March 2018

Molecular Analysis of Ephrin A4 and Ephrin B1 in a Rabbit Model of Craniosynostosis: Likely Exclusion as the Loci of Origin.

Cleft Palate Craniofac J 2018 08 22;55(7):1020-1025. Epub 2018 Feb 22.

Craniosynostosis (CS) has a prevalence of approximately 1 in every 2000 live births and is characterized by the premature fusion of one or more cranial sutures. Failure to maintain the cell lineage boundary at the coronal suture is thought to be involved in the pathology of some forms of CS. The Ephrin family of receptor tyrosine kinases consists of membrane-bound receptors and ligands that control cell patterning and the formation of developmental boundaries. Mutations in the ephrin A4 (EFNA4) and ephrin B1 (EFNB1) ligands have been linked to nonsyndromic CS and craniofrontonasal syndrome, respectively, in patient samples. We have previously described a colony of rabbits with a heritable pattern of coronal suture synostosis, although the genetic basis for synostosis within this model remains unknown. The present study was performed to determine if EFNA4 or EFNB1 could be the loci of the causal mutation in this unique animal model. Sequencing of EFNA4 and EFNB1 was performed using templates obtained from wild-type (n = 4) and craniosynostotic (n = 4) rabbits. No structural coding errors were identified in either gene. A single-nucleotide transversion was identified in one wild-type rabbit within the third intron of EFNA4. These data indicate that the causal locus for heritable CS in this rabbit model is not located within the structural coding regions of either EFNA4 or EFNB1.
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http://dx.doi.org/10.1597/16-135DOI Listing
August 2018

Simonart's Band: Its Effect on Cleft Classification and Recommendations for Standardized Nomenclature.

Cleft Palate Craniofac J 2017 11 12;54(6):726-733. Epub 2016 Sep 12.

Objective: Accurate classification of cleft lip plays an important role in communication, treatment planning, and comparison of outcomes across centers. Although there is reasonable consensus in defining cleft types, the presence of Simonart's band can make classification challenging. Our objective was to survey cleft care providers to determine what all consider to be Simonart's band, how its presence effects cleft lip classification, and to provide recommendations for standardized nomenclature.

Design: A multiple-choice survey was e-mailed to 1815 members of the American Cleft Palate-Craniofacial Association, assessing each respondent's definition of Simonart's band and its effect on cleft classification. Cleft classification was drawn from the ICD system diagnosis billing codes. Descriptive analysis was performed.

Results: Three hundred seventy-three providers completed the survey (20.5% response), the majority of whom were surgeons (61.5%); 87.1% agreed with the definition that a Simonart's band is "any soft tissue bridge located at the base of the nostril or more internally, between the segmented ridges." However, only 41.8% felt that the presence of a Simonart's band rendered a cleft lip incomplete; 54.4% felt that an alveolar cleft was the defining difference between a complete and an incomplete cleft lip. When asked to define the child with a cleft involving the upper lip that extends into the naris but interrupted by a soft tissue bridge located only at the base of the nostril or more internally, without a cleft of the alveolar ridge and palate, 61.4% classified this as an incomplete cleft lip, 32.7% as a complete cleft lip, and 5.9% as an unspecified cleft lip.

Conclusions: Responses revealed wide discrepancy in the classification of cleft phenotypes and in the interpretation of the significance of anatomical components in the classification of a cleft lip. We discuss the difficulty in aligning classification based on unclear definition of terms and variable anatomic parameters. We highlight this issue in the face of a need for comparability in clinical evidence-based practices. To ensure precision and uniformity in cleft classification, we recommend that use of the term "Simonart's band" be abandoned while incorporating a notation of the integrity of the nasal sill into the LAHSHAL system. We propose a uniform definition of incomplete versus complete cleft lip, wherein a cleft lip will be classified as complete in the presence or absence of narrow bands of tissue present at the base of the nasal sill or more internally.
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http://dx.doi.org/10.1597/15-319DOI Listing
November 2017

Rescue of Premature Coronal Suture Fusion with TGF-β2 Neutralizing Antibody in Rabbits with Delayed-Onset Synostosis.

Cleft Palate Craniofac J 2018 07 26;55(6):844-855. Epub 2018 Feb 26.

Objectives: An overexpression of Tgf-β2 leads to calvarial hyperostosis and suture fusion in individuals with craniosynostosis. Inhibition of Tgf-β2 may help rescue fusing sutures and restore normal growth. The present study was designed to test this hypothesis.

Design: Twenty-eight New Zealand White rabbits with delayed-onset coronal synostosis had radiopaque markers placed on either side of the coronal sutures at 10 days of age. The rabbits were randomly assigned to: (1) sham control rabbits (n = 10), (2) rabbits with control IgG (100 μg/suture) delivered in a collagen vehicle (n = 9), and (3) rabbits with Tgf-β2 neutralizing antibody (100 μg/suture) delivered in a collagen vehicle (n = 9). Longitudinal growth data were collected at 10, 25, 42, and 84 days of age. Sutures were harvested at 84 days of age for histomorphometry.

Results: Radiographic analysis showed significantly greater ( P < .05) coronal suture marker separation, craniofacial length, cranial vault length, height, shape indices, cranial base length, and more lordotic cranial base angles in rabbits treated with anti-Tgf-β2 antibody than in controls at 42 and 84 days of age. Histologically, rabbits treated with anti-Tgf-β2 antibody at 84 days of age had patent and significantly ( P < .05) wider coronal sutures and greater sutural area compared to controls.

Conclusions: These data support our hypothesis that antagonism of Tgf-β2 may rescue fusing coronal sutures and facilitate craniofacial growth in this rabbit model. These findings also suggest that cytokine therapy may have clinical significance in infants with progressive postgestational craniosynostosis.
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http://dx.doi.org/10.1597/16-065DOI Listing
July 2018

Application of Laser Capture Microdissection to Craniofacial Biology: Characterization of Anatomically Relevant Gene Expression in Normal and Craniosynostotic Rabbit Sutures.

Cleft Palate Craniofac J 2017 01 8;54(1):109-118. Epub 2016 Mar 8.

Objective: Fusion of the cranial sutures is thought to depend on signaling among perisutural tissues. Mapping regional variations in gene expression would improve current models of craniosynostosis. Laser capture microdissection (LCM) isolates discrete cell populations for gene expression analysis. LCM has rarely been used in the study of mineralized tissue. This study sought to evaluate the potential use of LCM for mapping of regional gene expression within the cranial suture.

Design: Coronal sutures were isolated from 10-day-old wild-type and craniosynostotic (CS) New Zealand White rabbits, and LCM was used to isolate RNA from the sutural ligament (SL), osteogenic fronts (OF), dura mater, and periosteum. Relative expression levels for Fibroblast Growth Factor 2 (FGF2), Fibroblast Growth Factor Receptor 2 (FGFR2), Transforming Growth Factor Beta 2 (TGFβ-2), Transforming Growth Factor Beta 3 (TGFβ-3), Bone Morphogenetic Protein 2 (BMP-2), Bone Morphogenetic Protein 4 (BMP-4), and Noggin were determined using quantitative real-time PCR.

Results: A fivefold increase in TGFβ2 expression was detected in the CS SL relative to wild type, whereas 152-fold less TGFβ-3 was detected within the OF of CS animals. Noggin expression was increased by 10-fold within the CS SL, but reduced by 13-fold within the CS dura. Reduced expression of FGF2 was observed within the CS SL and dura, whereas increased expression of FGFR2 was observed within the CS SL. Reduced expression of BMP-2 was observed in the CS periosteum, and elevated expression of BMP-4 was observed in the CS SL and dura.

Conclusions: LCM provides an effective tool for measuring regional variations in cranial suture gene expression. More precise measurements of regional gene expression with LCM may facilitate efforts to correlate gene expression with suture morphogenesis and pathophysiology.
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http://dx.doi.org/10.1597/15-114DOI Listing
January 2017

Genetic Homozygosity and Phenotypic Variability in Craniosynostotic Rabbits.

Cleft Palate Craniofac J 2017 01 16;54(1):94-99. Epub 2016 Feb 16.

Background: Craniosynostosis ranges in severity from single suture involvement with prenatal onset to multiple suture involvement with postnatal onset. The present study was designed to test the hypothesis that increasing homozygosity may be responsible for more severe phenotypic expression by examining the relationship between inbreeding and phenotypic expression in synostotic rabbits.

Methods: Data were obtained from 173 litters and 209 rabbits with familial craniosynostosis. Five distinct phenotypes were identified (normal n = 62; unicoronal delayed onset synostosis (DOS) n = 47; bicoronal DOS n = 21; unicoronal early onset synostosis (EOS) n = 26, and bicoronal EOS n= 53). Wright's coefficients of inbreeding (CI) were calculated using CompuPed software. Radiographs were taken at 10, 25, 42, 84, and 126 days of age to assess coronal suture, craniofacial, and skeletal growth. The relationship between CI and growth data was assessed using correlation coefficients.

Results: Mean CIs ranged from 15.68 (±2.22) in normal rabbits to 25.89 (±5.03) in bicoronal DOS, to 36.29 (±2.10) in unicoronal EOS to 42.85 (±2.10) in bicoronal EOS rabbits. Significant differences were noted among groups (F = 11.48; P < .001). Significant negative correlations were noted between CI and sutural and craniofacial growth at 25 (r = -.45, P < .001; and r = -.66, P < .001) through 126 (r = -.40, P < .001 and r = -.46, P < .001) days of age.

Conclusions: While the synostotic phenotype is inherited in an autosomal dominant fashion in these rabbits, increasing homozygosity is associated with more severely affected phenotypes. These findings suggest that an accumulation of additional, modifier genes may determine the severity of the synostotic phenotype in rabbits.
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http://dx.doi.org/10.1597/15-226DOI Listing
January 2017

Repair of a Complicated Calvarial Defect: Reconstruction of an Infected Wound With rhBMP-2.

Ann Plast Surg 2016 Feb;76(2):205-10

From the *Department of Plastic Surgery, University of Pittsburgh, Pittsburgh, PA; †Georgia Health Sciences University, Augusta, GA; ‡Division of Plastic Surgery, University of Wisconsin, Madison, WI; §Department of General Surgery, St Louis University, St Louis, MO; ∥Department of Oral Biology, and ¶Departments of Anthropology and Orthodontics, University of Pittsburgh, Pittsburgh, PA.

Background: Management of the previously infected craniofacial defect remains a significant clinical challenge, posing obstacles such as wound healing complications, lack of donor site availability, and predisposition to failure of the repair. Optimal therapy would reconstruct like with like, without donor site morbidity. The purpose of this study was to compare the efficacy of recombinant human bone morphogenetic protein-2 (rhBMP-2)-mediated bone regeneration with the current standard of autologous bone graft for repair of previously infected calvarial defects.

Methods: Nineteen adult New Zealand white rabbits underwent subtotal calvariectomy. Bone flaps were inoculated with Staphylococcus aureus and replanted. After 1 week of infection, bone flaps were removed, and wounds were debrided, followed by 10 days of antibiotic treatment. After 6 weeks, animals underwent scar debridement followed by definitive reconstruction in 1 of 4 groups: empty control (n = 3), vehicle control (buffer solution on absorbable collagen sponge [ACS], n = 3), autologous bone graft (n = 3), or rhBMP-2 repair (rhBMP-2/ACS, n = 10). Animals underwent computed tomography imaging at 0, 2, 4, and 6 weeks postoperatively, followed by euthanization and histological analysis. Percent healing was determined by 3-dimensional analysis. A (time × group) 2-way analysis of variance was performed on healing versus treatment group and postoperative time.

Results: At 6 weeks postoperatively, rhBMP-2/ACS and autologous bone graft resulted in 93% and 68% healing, respectively, whereas the empty and vehicle control treatment resulted in 27% and 26% healing (P < 0.001). Histologically, compared to autologous bone graft, bone in the rhBMP-2/ACS group was more cellular and more consistently continuous with wound margins.

Conclusions: The rhBMP-2 therapy is effective in achieving radiographic coverage of previously infected calvarial defects.
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http://dx.doi.org/10.1097/SAP.0000000000000515DOI Listing
February 2016

The influence of surgical correction on white matter microstructural integrity in rabbits with familial coronal suture craniosynostosis.

Neurosurg Focus 2015 May;38(5):E3

Department of Neurological Surgery, University of Pittsburgh Medical Center;

OBJECT Craniosynostosis is a condition in which one or more of the calvarial sutures fuses prematurely. In addition to the cosmetic ramifications attributable to premature suture fusion, aberrations in neurophysiological parameters are seen, which may result in more significant damage. This work examines the microstructural integrity of white matter, using diffusion tensor imaging (DTI) in a homogeneous strain of rabbits with simple, familial coronal suture synostosis before and after surgical correction. METHODS After diagnosis, rabbits were assigned to different groups: wild-type (WT), rabbits with early-onset complete fusion of the coronal suture (BC), and rabbits that had undergone surgical correction with suturectomy (BC-SU) at 10 days of age. Fixed rabbit heads were imaged at 12, 25, or 42 days of life using a 4.7-T, 40-cm bore Avance scanner with a 7.2-cm radiofrequency coil. For DTI, a 3D spin echo sequence was used with a diffusion gradient (b = 2000 sec/mm(2)) applied in 6 directions. RESULTS As age increased from 12 to 42 days, the DTI differences between WT and BC groups became more pronounced (p < 0.05, 1-way ANOVA), especially in the corpus callosum, cingulum, and fimbriae. Suturectomy resulted in rabbits with no significant differences compared with WT animals, as assessed by DTI of white matter tracts. Also, it was possible to predict to which group an animal belonged (WT, BC, and BC-SU) with high accuracy based on imaging data alone using a linear support vector machine classifier. The ability to predict to which group the animal belonged improved as the age of the animal increased (71% accurate at 12 days and 100% accurate at 42 days). CONCLUSIONS Craniosynostosis results in characteristic changes of major white matter tracts, with differences becoming more apparent as the age of the rabbits increases. Early suturectomy (at 10 days of life) appears to mitigate these differences.
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http://dx.doi.org/10.3171/2015.2.FOCUS14849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347148PMC
May 2015

Transforming growth factor beta 1 augments calvarial defect healing and promotes suture regeneration.

Tissue Eng Part A 2015 Mar 6;21(5-6):939-47. Epub 2015 Feb 6.

1 Department of Plastic Surgery, University of Pittsburgh , Pittsburgh, Pennsylvania.

Background: Repair of complex cranial defects is hindered by a paucity of appropriate donor tissue. Bone morphogenetic protein 2 (BMP2) and transforming growth factor beta 1 (TGFβ1) have been shown separately to induce bone formation through physiologically distinct mechanisms and potentially improve surgical outcome for cranial defect repair by obviating the need for donor tissue. We hypothesize that a combination of BMP2 and TGFβ1 would improve calvarial defect healing by augmenting physiologic osteogenic mechanisms.

Methods/results: Coronal suturectomies (3×15 mm) were performed in 10-day-old New Zealand White rabbits. DermaMatrix™ (3×15mm) patterned with four treatments (vehicle, 350 ng BMP2, 200 ng TGFβ1, or 350 ng BMP2+200 ng TGFβ1) was placed in suturectomy sites and rabbits were euthanized at 6 weeks of age. Two-dimensional (2D) defect healing, bone volume, and bone density were quantified by computed tomography. Regenerated bone was qualitatively assessed histologically. One-way analysis of variance revealed significant group main effects for all bone quantity measures. Analysis revealed significant differences in 2D defect healing, bone volume, and bone density between the control group and all treatment groups, but no significant differences were detected among the three growth factor treatment groups. Qualitatively, TGFβ1 treatment produced bone with morphology most similar to native bone. TGFβ1-regenerated bone contained a suture-like tissue, growing from the lateral edge of the defect margin toward the midline. Unique to the BMP2 treatment group, regenerated bone contained lacunae with chondrocytes, demonstrating the presence of endochondral ossification.

Conclusions/significance: Total healing in BMP2 and TGFβ1 treatment groups is not significantly different. The combination of BMP2+TGFβ1 did not significantly increase bone healing compared with treatment with BMP2 or TGFβ1 alone postoperatively at 4 weeks. We highlight the potential use of TGFβ1 to regenerate calvarial bone and cranial sutures. TGFβ1 therapy significantly augmented bony defect healing at an earlier time point when compared with control, regenerated bone along the native intramembranous ossification pathway, and (unlike BMP2 alone or in combination with TGFβ1) permitted normal suture reformation. We propose a novel method of craniofacial bone regeneration using low-dose, spatially controlled growth factor therapies to minimize potentially harmful effects while maximizing local bioavailability and regenerating native tissues.
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http://dx.doi.org/10.1089/ten.TEA.2014.0189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4356478PMC
March 2015

Bone morphogenetic protein 2–mediated mandible reconstruction successfully heals bony defects but inhibits concurrent inferior alveolar nerve grafting: a rabbit experimental model.

J Craniofac Surg 2014 Nov;25(6):2241-5

Background: Bone morphogenetic protein 2 (BMP-2) has been used to reconstruct mandibular defects. An elegant addition to this reconstruction method would be incorporation of a nerve graft wrapped in a BMP-2 carrier to reconstitute the inferior alveolar nerve (IAN) and restore sensation to the lower face. We developed a rabbit model to determine the effect BMP-2 has on nerve regeneration following neurorrhaphy.

Methods: An inferior border mandibulectomy was created in 16 adult New Zealand white rabbits. The IAN was protected, divided, and repaired with either primary neurorrhaphy or reverse autografts. Bone defects were treated with no treatment controls (n = 2), absorbable collagen sponge (ACS) (vehicle controls) (n = 7), and ACS soaked in BMP-2 (treatment group) (n = 7). Animals underwent computed tomography (CT) 2 days and 6 weeks postoperatively. The percent bone defect healing was calculated using Amira 3D imaging software. At 6 weeks, IANs were harvested mesial to the reconstruction and were evaluated with toluidine blue histology to identify myelinated axons. Reconstructed mandible segments were evaluated with micro-CT and hematoxylin-eosin histology.

Results: Bone morphogenetic protein 2-treated animals demonstrated significantly more bone healing than did the ACS and empty defect groups (82%, 38%, 44%, respectively; P < 0.01). One hundred percent of ACS-treated nerves (n = 4) demonstrated axon regrowth, whereas only 25% of BMP-2-treated nerves (n = 4) did. Micro-CT and histology showed BMP-2 caused bone growth around the IAN, but regenerated bone infiltrated the repair site and created a physical barrier to axon growth.

Conclusions: Bone morphogenetic protein 2 can successfully heal bone defects in the rabbit mandible, but ectopic bone growth can inhibit IAN recovery after repair. Level of Evidence: Not gradable.
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http://dx.doi.org/10.1097/SCS.0000000000001051DOI Listing
November 2014

Cleft Palate-Craniofacial Journal 50th anniversary editorial board commentary: anatomy, basic sciences, and genetics--then and now.

Cleft Palate Craniofac J 2014 May 11;51(3):253-6. Epub 2014 Mar 11.

To celebrate the 50th year of the Cleft Palate-Craniofacial Journal we look back to where we started in 1964 and where we are now, and we speculate about directions for the future in a "Then and Now" editorial series. This editorial examines changing trends and perspectives in anatomical, basic science, and genetic studies published in this 50-year interval. In volume 1 there were 45 total papers, seven (16%) of which were peer-reviewed basic science and genetic articles published: four in anatomy, three in craniofacial biology, and none in genetics. In contrast, in volume 50, of 113 articles there were 47 (42%) peer-reviewed basic science and genetic articles published: 30 in anatomy, five in craniofacial biology, and 12 in genetics. Topical analysis of published manuscripts then and now reveal that similar topics in anatomy and craniofacial biology are still being researched today (e.g., phenotypic variability, optimal timing of surgery, presurgical orthopedics, bone grafting); whereas, most of the more recent papers use advanced technology to address old questions. In contrast, genetic publications have clearly increased in frequency during the last 50 years, which parallels advances in the field during this time. However, all of us have noticed that the more "cutting-edge" papers in these areas are not being submitted for publication to the journal, but instead to discipline-specific journals. Concerted efforts are therefore indicated to attract and publish these cutting-edge papers in order to keep the Cleft Palate-Craniofacial Journal in the forefront of orofacial cleft and craniofacial anomaly research and to provide a valuable service to American Cleft Palate-Craniofacial Association members.
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http://dx.doi.org/10.1597/14-022DOI Listing
May 2014

Ectocranial suture fusion in primates: pattern and phylogeny.

J Morphol 2014 Mar 21;275(3):342-7. Epub 2013 Oct 21.

Departments of Oral Biology, Orthodontics, Cellular Biology and Anatomy, Orthopaedic Surgery, and Surgery-Plastic Surgery, Georgia Regents University, Augusta, Georgia.

Patterns of ectocranial suture fusion among Primates are subject to species-specific variation. In this study, we used Guttman Scaling to compare modal progression of ectocranial suture fusion among Hominidae (Homo, Pan, Gorilla, and Pongo), Hylobates, and Cercopithecidae (Macaca and Papio) groups. Our hypothesis is that suture fusion patterns should reflect their evolutionary relationship. For the lateral-anterior suture sites there appear to be three major patterns of fusion, one shared by Homo-Pan-Gorilla, anterior to posterior; one shared by Pongo and Hylobates, superior to inferior; and one shared by Cercopithecidae, posterior to anterior. For the vault suture pattern, the Hominidae groups reflect the known phylogeny. The data for Hylobates and Cercopithecidae groups is less clear. The vault suture site termination pattern of Papio is similar to that reported for Gorilla and Pongo. Thus, it may be that some suture sites are under larger genetic influence for patterns of fusion, while others are influenced by environmental/biomechanic influences.
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http://dx.doi.org/10.1002/jmor.20218DOI Listing
March 2014

Cloning of TgfβR1 and TgfβR2 and Likely Exclusion as Loci of Origin in a Rabbit Craniosynostotic Model.

Cleft Palate Craniofac J 2014 Jan 12;51(1):56-69. Epub 2013 Jun 12.

Objective: To determine whether TgfβR1 or TgfβR2 cause the craniosynostotic phenotype in a rabbit model of nonsyndromic craniosynostosis.

Design: Full-length TgfβR1 and TgfβR2 cDNAs were sequenced and real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed to measure TgfβR1 and TgfβR2 transcripts in sutural tissue from wild type (WT) and craniosynostotic (CS) rabbits. Single nucleotide polymorphisms (SNP) were identified within TgfβR1 and TgfβR2 and were assayed for segregation with disease phenotype in 22 craniosynostotic animals.

Results: No structural mutations in TgfβR1 and TgfβR2 were identified in the craniosynostotic rabbits. Real-time RT-PCR quantification of TgfβR1 and TgfβR2 mRNA showed no significant difference in TgfβR1 expression between CS and WT animals, while TgfβR2 showed 50% elevation in the CS animals compared to WT (P < .05). SNP analysis within the TgfβR1 and TgfβR2 genes suggested that neither locus is linked to the craniosynostotic phenotype because no allelic combination showed any specific correlation with disease phenotype for either TgfβR1 or TgfβR2.

Conclusions: Our data indicate that the craniosynostotic phenotype in this rabbit model does not arise from any structural mutation in TgfβR1 or TgfβR2, and SNP analysis also likely excludes these genes more broadly as the site of causative mutation.
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http://dx.doi.org/10.1597/12-160DOI Listing
January 2014

Molecular Analysis of Twist1 and FGF Receptors in a Rabbit Model of Craniosynostosis: Likely Exclusion as the Loci of Origin.

Int J Genomics 2013 8;2013:305971. Epub 2013 May 8.

Department of Plastic Surgery, University of Pittsburgh, Pittsburgh, PA 15261, USA.

Craniosynostosis is the premature fusion of the cranial vault sutures. We have previously described a colony of rabbits with a heritable pattern of nonsyndromic, coronal suture synostosis; however, the underlying genetic defect remains unknown. We now report a molecular analysis to determine if four genes implicated in human craniosynostosis, TWIST1 and fibroblast growth factor receptors 1-3 (FGFR1-3), could be the loci of the causative mutation in this unique rabbit model. Single nucleotide polymorphisms (SNPs) were identified within the Twist1, FGFR1, and FGFR2 genes, and the allelic patterns of these silent mutations were examined in 22 craniosynostotic rabbits. SNP analysis of the Twist1, FGFR1, and FGFR2 genes indicated that none were the locus of origin of the craniosynostotic phenotype. In addition, no structural mutations were identified by direct sequence analysis of Twist1 and FGFR3 cDNAs. These data indicate that the causative locus for heritable craniosynostosis in this rabbit model is not within the Twist1, FGFR1, and FGFR2 genes. Although a locus in intronic or flanking sequences of FGFR3 remains possible, no direct structural mutation was identified for FGFR3.
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http://dx.doi.org/10.1155/2013/305971DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3664496PMC
June 2013

Novel animal model of calvarial defect: part IV. Reconstruction of a calvarial wound complicated by durectomy.

Plast Reconstr Surg 2013 Apr;131(4):512e-519e

Pittsburgh, Pa.; and Augusta, Ga. From the Departments of Plastic Surgery, Oral Biology, Anthropology, Orthodontics, and Bioengineering, University of Pittsburgh, and the Georgia Health Sciences University.

Background: Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been shown to be an effective therapy in the acute calvarial defect wound and in calvarial defects complicated by chronic scar and radiation. The authors assessed the effectiveness of rhBMP-2-mediated bone regeneration in calvarial defects complicated by durectomy.

Methods: Sixteen adult New Zealand White rabbits underwent subtotal calvariectomy and dural removal, followed by dural repair and reconstruction in one of four groups: empty (n = 3), vehicle (buffer solution on an absorbable collagen sponge, n = 2), autologous graft (n = 3), or rhBMP-2 repair (rhBMP-2/absorbable collagen sponge, n = 8). Animals underwent computed tomographic imaging at 0, 2, 4, and 6 weeks postoperatively, followed by euthanasia and histologic analysis. Percent healing was determined by three-dimensional analysis. A 4 × 3 mixed model analysis of variance was performed on healing versus treatment group/postoperative time.

Results: The rhBMP-2/absorbable collagen sponge and autograft repair groups had 51.4 and 37.3 percent healing, respectively, at 6 weeks; empty and vehicle control groups had 7.8 and 17.9 percent healing, respectively, at 6 weeks. Compared with immediate favorable reconstruction (96.8 percent healing), rhBMP-2 in this setting was significantly less effective (p = 0.001). Bone in the rhBMP-2/absorbable collagen sponge group was compact and cellular but appeared only over the intact sagittal sinus and irregularly within the absorbable collagen sponge.

Conclusions: Although promising in the acute calvarial wound and other complex defects, rhBMP-2 therapy is less effective in reconstruction following dural compromise. Future studies using additional growth factors and cell therapy may improve results in this especially difficult scenario.
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http://dx.doi.org/10.1097/PRS.0b013e3182818b4cDOI Listing
April 2013

Novel animal model of calvarial defect: part III. Reconstruction of an irradiated wound with rhBMP-2.

Plast Reconstr Surg 2012 Nov;130(5):643e-650e

Pittsburgh, Pa. From the Division of Plastic Surgery and the Departments of Oral Biology, Anthropology, Orthodontics, and Bioengineering, University of Pittsburgh.

Background: Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been shown to be an effective therapy in the acute calvarial defect wound and in calvarial defects complicated by chronic scar. The authors compared the effectiveness of rhBMP-2 with the accepted standard of autologous graft for repair of irradiated calvarial defects.

Methods: Nineteen adult New Zealand White rabbits underwent subtotal calvariectomy. Four days postoperatively, animals received 15 Gy to their wound. Six weeks postoperatively, scars were débrided and defects reconstructed in one of four groups: empty (n = 3), vehicle (buffer solution/absorbable collagen sponge; n = 3), cryopreserved autograft, (n = 3), or rhBMP-2 repair (rhBMP-2/absorbable collagen sponge, n = 10). Animals underwent computed tomography imaging at 0, 2, 4, and 6 weeks, followed by euthanization and histological analysis. Percent healing was determined and a 4 × 3 mixed model analysis of variance was performed on healing versus treatment group/postoperative time.

Results: According to radiopacity, rhBMP-2/sponge and autografts were statistically equivalent, with 99 and 89 percent healing at 6 weeks. Empty and vehicle treatment groups, with 35 and 34 percent healing, were inferior to the rhBMP-2/sponge and autograft groups (p < 0.05). Histologically, bone in the surgical control (autograft) group was less cellular and trabecular than bone formed after rhBMP-2/sponge treatment.

Conclusions: rhBMP-2 therapy was as effective in reconstructing calvarial defects in the unfavorable irradiated wound as in the acute, favorable calvarial wound. Compared with cryopreserved autologous graft, rhBMP-2-regenerated bone resulted in equal defect coverage, similar thickness, and greater cellularity. Further studies are necessary to demonstrate the long-term viability and remodeling rhBMP-2/sponge-generated bone.
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http://dx.doi.org/10.1097/PRS.0b013e318267d412DOI Listing
November 2012

Ectocranial suture fusion in primates: as related to cranial volume and dental eruption.

J Med Primatol 2012 Dec 3;41(6):356-63. Epub 2012 Oct 3.

Departments of Oral Biology, Orthodontics, Surgery-Plastic Surgery and Graduate Studies, Georgia Health Sciences University, Augusta, GA, USA.

Background: Timing of calvarial suture fusion is important in primate ontogeny. Ages at death are difficult to assess especially for museum collections.

Methods: 1550 skulls of Hominoid, Hylobatidae, Macaca and Papio were observed for fusion. Calvarial expansion (early) and dental eruption (late) were utilized as indicators of ontogeny. Homogeneity of slopes and ANOVA were used to determine differences in timing of fusion.

Results: For calvarial growth the great apes all showed small levels of calvarial suture remodeling prior to full calvarial expansion. For dental eruption, Homo and Macaca share a common pattern of fusion in late adulthood. The other species show early remodeling. Papio was observed to have distinct patterns for suture fusion progression.

Conclusions: Thus, suture fusion progression although influenced by evolutionary changes in the robusticity of the craniofacial skeleton can be modeled by the phylogeny among this group. Overall, Homo appears to have a distinct pattern of delayed suture fusion progression.
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http://dx.doi.org/10.1111/jmp.12018DOI Listing
December 2012

Tissue interactions between craniosynostotic dura mater and bone.

J Craniofac Surg 2012 May;23(3):919-24

Division of Plastic and Reconstructive Surgery, Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15224, USA.

Background: Cells within the dura mater have been implicated in the determination of suture patency and fusion. Craniosynostosis (CS), the premature fusion of 1 or more of the cranial sutures, could result from abnormal control over the differentiation of osteoprogenitor cells from the dura mater. This study tested whether dura mater cells derived from rabbits with congenital CS were different from cells derived from normal rabbits and investigated the effects that CS dura mater had on osteogenic differentiation in vitro and in vivo.

Methods: Cells were derived from the dura mater from wild-type rabbits (WT; n = 23) or CS rabbits (n = 16). Cells were stimulated with bone morphogenetic protein 4, and alkaline phosphatase (ALP) expression and cell proliferation were assessed. Dura mater-derived cells were also cocultured with primary rabbit bone-derived cells, and ALP was assessed. Finally, interactions between the dura mater and overlying tissues were manipulated in vivo.

Results: Craniosynostotic dura mater-derived cells proliferated faster than did WT cells but were not more ALP positive. Coculture experiments showed that CS dura mater cells induced increased ALP activity in CS bone-derived cells, but not in WT bone-derived cells. In vivo experiments showed that a physical barrier successfully inhibited dura mater-derived osteogenesis.

Conclusions: Coculture of CS bone- and CS dura mater-derived cells evoked an abnormal phenotype in vitro. Covering the CS dura mater led to decreased bone formation in vivo. Further investigations will focus on the signaling molecules involved in the communication between these 2 CS tissue types in vitro and in vivo.
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http://dx.doi.org/10.1097/SCS.0b013e31824e645fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3360881PMC
May 2012

Novel model of calvarial defect in an infected unfavorable wound: reconstruction with rhBMP-2. Part II.

J Craniofac Surg 2012 Mar;23(2):410-4

Division of Plastic Surgery, University of Pittsburgh, Pittsburgh, PA 15201, USA.

Background: Animal models of bone reconstruction have shown recombinant human bone morphogenetic protein 2 (rhBMP-2) to be an effective therapy in the acute calvarial defect wound. The purpose of this study was to compare the effectiveness of rhBMP-2 in a rabbit model of an unfavorable scarred calvarial wound with the criterion standard of autograft.

Methods: Nineteen adult New Zealand white rabbits underwent subtotal calvariectomy. After 6 weeks of healing and normal scar formation, these animals underwent reoperation for scar debridement and assignment to 1 of 4 therapeutic groups. Animals were assigned to an empty control group (no treatment, n = 3), vehicle control group (neutral buffered solution on an absorbable collagen sponge [ACS], n = 3), surgical control group (cryopreserved autograft, n = 3), or an experimental treatment group (rhBMP-2 on an ACS, n = 10). All animals underwent computed tomography imaging at 0, 2, 4, and 6 weeks after secondary reconstructive surgery. At 6 weeks, all animals were killed, and the defects were examined histologically. Percentage of healing of each defect was determined, and a 4 × 3 mixed-model analysis of variance was performed on healing as a function of time and therapy.

Results: Based on measures of defect radiopacity, the treatment group (rhBMP-2/ACS) and surgical control group (autograft) were statistically equivalent with 98% and 83% healing, respectively, at 6 weeks. The empty control and vehicle control groups were inferior to the treatment group (rhBMP-2/ACS) and surgical control (autograft) groups at each timepoint (P < 0.05). Histologically, bone in the surgical control (autograft) group was less trabecular and less cellular than the bone formed in the experimental treatment group (rhBMP-2/ACS).

Conclusions: Compared with historical controls, rhBMP-2 therapy was as effective in reconstructing calvarial defects in the unfavorable scarred wound as in the acute favorable calvarial wound. When compared with cryopreserved autograft, rhBMP-2-regenerated bone showed equal defect coverage and similar bone thickness with varying bony architecture.
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http://dx.doi.org/10.1097/SCS.0b013e318240feb8DOI Listing
March 2012

Regenerative surgery in cranioplasty revisited: the role of adipose-derived stem cells and BMP-2.

Plast Reconstr Surg 2011 Nov;128(5):1053-1060

Pittsburgh, Pa. From the Division of Plastic Surgery, University of Pittsburgh.

Background: Reconstruction of the pediatric calvaria is frequently complicated by a shortage of bone. This problem is most apparent between 2 and 10 years of age, when the osteogenic potential of the dura is diminished and the diploic space has not matured to the point that split-thickness calvarial grafting is practical. In this article, the authors evaluate and compare the relative efficacy of adipose-derived stem cells, bone morphogenetic protein (BMP)-2, and adipose-derived stem cells osteoinduced with BMP-2 in addressing these defects.

Methods: Cranial defects measuring 15×15 mm were created in New Zealand White rabbits. Five treatment modalities were compared: no repair (surgical control); untreated acellular collagen sponge (vehicle control); BMP-2 on acellular collagen sponge; adipose-derived stem cells on acellular collagen sponge; and osteoinduced adipose-derived stem cells on acellular collagen sponge. Osteogenesis was assessed with radiology and histology. Statistical significance was determined by analysis of variance.

Results: No significant difference in osseous healing was observed among empty controls (32.8 percent), acellular collagen sponge alone (34.4 percent), adipose-derived stem cells on acellular collagen sponge (33.9 percent), and osteoinduced adipose-derived stem cells on acellular collagen sponge (40.2 percent). Defects reconstructed with recombinant human BMP-2/acellular collagen sponge were on average 96.9 percent ossified, significantly (p<0.05) more than the defects in all other groups.

Conclusions: BMP-2-based tissue engineering is a viable approach to craniofacial reconstruction. Adipose-derived stem cells did not significantly augment this process as modeled here. Advances in the understanding of craniofacial biology, and of protein- and cell-based therapies, will enhance the efficacy of tissue-engineering strategies for this problem in the future.
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http://dx.doi.org/10.1097/PRS.0b013e31822b65e4DOI Listing
November 2011

Relaxin does not rescue coronal suture fusion in craniosynostotic rabbits.

Cleft Palate Craniofac J 2012 Sep 8;49(5):e46-54. Epub 2011 Jul 8.

Department of Surgery, Division of Plastic Surgery, Pediatric Craniofacial Biology Laboratory, Children's Hospital of Pittsburgh, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

Objectives: Craniosynostosis affects 1 in 2000 to 3000 live births and may result in craniofacial and neural growth disturbances. Histological data have shown that thick collagenous bundles are present in the sutural ligament, which may tether the osteogenic fronts, resulting in premature fusion. The hormone relaxin has been shown to disrupt collagen fiber organization, possibly preventing craniosynostosis by relaxing the sutural ligament and allowing osteogenic fronts to separate normally and stay patent. This study tested this hypothesis with a rabbit model of delayed-onset coronal suture synostosis.

Methods: A total of 18 New Zealand White rabbits with craniosynostosis were randomly assigned to one of three groups: sham control, protein control (BSA), relaxin treatment. After initial diagnosis, sham surgery, BSA, or relaxin was delivered to the fusing coronal suture in a slow-release (56-day) collagen vehicle. Longitudinal radiographs and body weights were collected at 10, 25, 42, and 84 days of age, and sutures were harvested for histology.

Results: Relaxin-treated animals had more disorganized intrasuture content than control groups. These specimens also appeared to have relatively wider sutures ectocranially. There were no significant differences in relaxin-treated animals for all craniofacial growth measures, or suture separation compared with controls.

Conclusions: These data do not support our initial hypothesis that the use of relaxin may rescue sutures destined to undergo premature suture fusion. These findings suggest that collagen fiber arrangement may not be important for suture fusion. This protein therapy would not be clinically useful for craniosynostosis.
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http://dx.doi.org/10.1597/11-024DOI Listing
September 2012

Molecular analysis of coronal perisutural tissues in a craniosynostotic rabbit model using polymerase chain reaction suppression subtractive hybridization.

Plast Reconstr Surg 2011 Jul;128(1):95-103

Pittsburgh and Philadelphia, Pa. From the Department of Surgery, Division of Plastic and Reconstructive Surgery, University of Pittsburgh and Pediatric Craniofacial Biology Laboratory, Children's Hospital of Pittsburgh; the Center for Genomic Sciences, Allegheny-Singer Research Institute, West Penn Allegheny Health Systems; the Departments of Anthropology, Orthodontics, Oral Biology, and Bioengineering, University of Pittsburgh; the Division of Plastic Surgery, Allegheny General Hospital of Pittsburgh; and the Department of Microbiology and Immunology, Drexel University College of Medicine.

Background: In the United States, the incidence of craniosynostosis (premature fusion of the sutures of the cranial vault) is one in 2000 to 3000 live births. The condition can cause increased intracranial pressure, severely altered head shape, and mental retardation. The authors have previously described a colony of rabbits with heritable coronal suture synostosis. This model has been instrumental in describing the postsurgical craniofacial growth associated with craniosynostosis. The molecular analysis of this model has been limited by the lack of molecular tools for use in rabbits. To understand the pathogenesis of craniosynostosis, the authors compared gene expression in perisutural tissues between wild-type and craniosynostotic rabbits using polymerase chain reaction suppression subtractive hybridization.

Methods: Suppression subtractive hybridization polymerase chain reaction was performed on RNA derived from pooled samples of calvariae from 10-day-old wild-type (n = 3) and craniosynostotic (n = 3) rabbits to obtain cDNA clones enriched in either wild-type tissues (underexpressed in craniosynostotic tissue) or craniosynostotic tissues (overexpressed in craniosynostotic compared with wild-type).

Results: Differential expression was identified for approximately 140 recovered cDNA clones up-regulated in craniosynostotic tissues and 130 recovered clones for wild-type tissues. Of these, four genes were confirmed by quantitative reverse-transcriptase polymerase chain reaction as being overexpressed in craniosynostotic sutural tissue: β-globin (HBB), osteopontin (SPP1), osteonectin (SPARC), and cathepsin K (CTSK). Two genes were confirmed to be underexpressed in the craniosynostotic samples: collagen 3, alpha 1 (COL3A1) and ring finger protein 12 (RNF12).

Conclusion: The differential expression of these gene products in our naturally occurring craniosynostotic model appears to be the result of differences in the normal bone formation/resorption pathway.
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http://dx.doi.org/10.1097/PRS.0b013e31821740e8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3563161PMC
July 2011

Masticatory hypermuscularity is not related to reduced cranial volume in myostatin-knockout mice.

Anat Rec (Hoboken) 2011 Jul 25;294(7):1170-7. Epub 2011 May 25.

Division of Plastic and Reconstructive Surgery, Department of Surgery, University of Pittsburgh, Pennsylvania 15201, USA.

It has been suggested recently that masticatory muscle size reduction in humans resulted in greater encephalization through decreased compressive forces on the cranial vault. Following this logic, if masticatory muscle size were increased, then a reduction in brain growth should also occur. This study was designed to test this hypothesis using a myostatin (GDF-8) knockout mouse model. Myostatin is a negative regulator of skeletal muscle growth, and individuals lacking this gene show significant hypermuscularity. Sixty-two [32 wild-type (WT) and 30 GDF-8 -/- knockout], 1, 28, 56, and 180-day-old CD-1 mice were used. Body and masseter muscle weights were collected following dissection and standardized lateral and dorsoventral cephalographs were obtained. Cephalometric landmarks were identified on the radiographs and cranial volume was calculated. Mean differences were assessed using a two-way ANOVA. KO mice had significantly greater body and masseter weights beginning at 28 days compared with WT controls. No significant differences in cranial volumes were noted between KO and WT. Muscle weight was not significantly correlated with cranial volume in 1, 28, or 180-day-old mice. Muscle weights exhibited a positive correlation with cranial volume at 56 days. Results demonstrate that masticatory hypermuscularity is not associated with reduced cranial volume. In contrast, there is abundant data demonstrating the opposite, brain growth determines cranial vault growth and masticatory apparatus only affects ectocranial morphology. The results presented here do not support the hypothesis that a reduction in masticatory musculature relaxed compressive forces on the cranial vault allowing for greater encephalization.
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http://dx.doi.org/10.1002/ar.21412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3791924PMC
July 2011

BMP-2-mediated regeneration of large-scale cranial defects in the canine: an examination of different carriers.

Plast Reconstr Surg 2011 May;127(5):1865-1873

Pittsburgh, Pa. From the Division of Plastic Surgery and the Departments of Oral Biology, Anthropology, Orthodontics, and Bioengineering, University of Pittsburgh.

Background: Recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered on an absorbable collagen sponge is a U.S. Food and Drug Administration-approved therapy shown to be an effective means of generating bone formation in multiple clinical settings. However, the optimum dose and delivery of rhBMP-2 to the calvaria are undetermined. The aim of the authors' study was to investigate the use of rhBMP-2 in addressing calvarial defects in a large-animal model through a variety of modifications to this U.S. Food and Drug Administration-approved therapy.

Methods: Twenty-three adult canines underwent the creation of a standard calvarial defect and received either no treatment, 0.2 mg/ml rhBMP-2 in an absorbable collagen sponge, 0.2 mg/ml rhBMP-2 in an absorbable collagen sponge with corticocancellous chips, 0.2 mg/ml rhBMP-2 in an absorbable collagen sponge with MasterGraft Granules, or 0.4 mg/ml rhBMP-2 in a compression-resistant matrix carrier. Direct comparisons of defect radiopacity were performed at 0, 8, 16, and 24 weeks postoperatively before the animals were euthanized. All specimens were evaluated qualitatively with histology.

Results: Surgical control animals had an average defect radiopacity of 32.7 percent at study completion compared with an average of 99.95 percent across all treatment groups. Ectopic bone formation was found consistently in all treatment groups with varying degrees of severity. Regenerated bone thickness, compactness, and organization varied qualitatively between groups.

Conclusions: Treatment with 0.2 mg/ml rhBMP-2 in an absorbable collagen sponge with MasterGraft Granules showed the least amount of ectopic bone formation and the most compact bone formation within the defect, and produced reasonably consistent bony thickness across the defect. Future studies should focus on spatial regulation of rhBMP-2 to minimize unwanted bone formation.
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http://dx.doi.org/10.1097/PRS.0b013e31820cf2c9DOI Listing
May 2011