Jack C. Yu

Age-Related Changes in the Osteogenic Differentiation Potential of Mouse Bone Marrow Stromal Cells

Age‐dependent bone loss has been well documented in both human and animal models. Although the underlying causal mechanisms are probably multifactorial, it has been hypothesized that alterations in progenitor cell number or function are important. Little is known regarding the properties of bone marrow stromal cells (BMSCs) or bone progenitor cells during the aging process, so the question of whether aging alters BMSC/progenitor osteogenic differentiation remains unanswered. In this study, we examined age‐dependent changes in bone marrow progenitor cell number and differentiation potential between mature (3 and 6 mo old), middle‐aged (12 and 18 mo old), and aged (24 mo old) C57BL/6 mice. BMSCs or progenitors were isolated from five age groups of C57BL/6 mice using negative immunodepletion and positive immunoselection approaches. The osteogenic differentiation potential of multipotent BMSCs was determined using standard osteogenic differentiation procedures. Our results show that both BMSC/progenitor number and differentiation potential increase between the ages of 3 and 18 mo and decrease rapidly thereafter with advancing age. These results are consistent with the changes of the mRNA levels of osteoblast lineage‐associated genes. Our data suggest that the decline in BMSC number and osteogenic differentiation capacity are important factors contributing to age‐related bone loss.

Speech characteristics associated with the Furlow palatoplasty as compared with other surgical techniques.

&NA; Reported here are the results of a retrospective study of the speech outcome for 63 cleft subjects who had Furlow repairs compared with 20 subjects who had other procedures. The two groups of children were similar in cleft type, sex, and race. The same two surgeons repaired the palates in both groups, and the same two speech pathologists with high reliability examined the children at least 5 years postoperatively using the Pittsburgh Weighted Values for Speech Symptoms Associated with VPI (velopharyngeal incompetence). Subjects who had had Furlow repairs were superior on measures of hypernasality, articulation, and total speech scores; and fewer pharyngeal flaps were required by Furlow subjects. These findings suggest the need for randomized, double‐blind investigations comparing outcome of the Furlow procedure with the intravelar veloplasty, the V‐Y pushback, and other specified procedures.

The diagnosis and treatment of single-sutural synostoses : Are computed tomographic scans necessary?

Background: Computed tomographic scan evaluation is the current standard of care for diagnosing craniosynostosis. Recent publications, and the National Cancer Institute, have raised concerns about ionizing radiation associated with computed tomographic scans in children (e.g., developmental delays, tumor induction). The authors sought to ascertain the diagnostic accuracy of the physical examination in evaluating single-sutural craniosynostosis and assess the need for computed tomographic scans in surgical correction. Methods: This prospective, multicenter, outcome assessment included children clinically diagnosed with a single-sutural synostosis by craniofacial surgeons (with 1 to 18 years’ experience) at four centers over a 1-year period. Clinical diagnoses were compared with computed tomographic scan evaluations. All surgeons scored the utility of computed tomographic scans during surgical repair. Results: Sixty-seven patients were clinically diagnosed with single-sutural craniosynostosis (mean age, 7 months; range, 1 week to 48 months). Sixty-six of 67 patients were diagnosed with craniosynostosis by computed tomographic scan (sagittal, 40 percent; metopic, 31 percent; right unilateral coronal, 16 percent; left unilateral coronal, 6 percent; and right lambdoid, 6 percent), for a diagnostic accuracy exceeding 98 percent. One patient with suspected lambdoid synostosis was radiologically diagnosed with positional plagiocephaly. Three of four craniofacial surgeons scored computed tomographic scans as “unnecessary” for surgical correction; one scored scans as “sometimes helpful.” Conclusions: Craniofacial surgeons with various experience levels were able to accurately diagnose single-sutural synostosis by physical examination alone. Considering potential side effects from ionizing radiation, risks of sedation, and costs, surgeons may wish to reserve computed tomographic scans only for infants with suspected single-sutural craniosynostosis in whom the physical examination is not clearly diagnostic.

Extrinsic tension results in FGF-2 release, membrane permeability change, and intracellular Ca++ increase in immature cranial sutures.

There are numerous studies cataloging the temporal profiles of the various growth factors during the morphogenesis of cranial sutures. There are also many clearly documented mutations of the receptors of some of these growth factors such as fibroblast growth factor (FGF)R-2 and FGFR-3 in clinical craniosynostosis. It is obvious, and often concluded, that growth factors play a role or are involved in craniofacial development. However, precisely what that role is, what causes the changes in the growth factor levels, and why these changes occur in the particular temporal and spatial patterns observed remains elusive. Using simple physics, we applied a plasma membrane disruption model and the principles of complex adaptive systems to arrive at a conjecture of calvarial morphogenesis. The purpose of this article is to introduce the concept of complex adaptive systems, to propose our conjecture, and to provide experimental proof of some key steps in this conjecture: tension induces rapid and demonstrable physiological responses in some cells within the immature cranial sutures. These responses include increases of intracellular Ca ++, plasma membrane permeability, and the release of growth factors, e.g., FGF-2. Paired coronal sutures from 1-week-old Sprague-Dawley rat pups were subjected to either 0.59 N of tensile force or no force for 5 minutes in a protein-free medium. FGF-2 levels in the media were measured by slot blot analysis. Western blot analysis was used to determine FGF-2 levels in the sutures. To determine cell membrane permeability changes, fluorescein-conjugated dextran, with a molecular weight of 10 kd, was added to the media during the 5 minutes with or without tensile force. Laser confocal microscopy was used to compare the amount of entry of this impermeant tracer and the pattern of permeability change at the tissue level. To determine the intracellular pCa ++, the sutures were first loaded with a calcium indictor, FURA-2 AM, and then subjected isotonically to 0.059 N of tension. The intracellular pCa ++ was expressed as ratio of Ca ++ -bound FURA-2 to Ca ++ -free FURA-2. The experimental findings were as follows: 1) Sutures, in response to tension, release FGF-2. 2) Sutures contain higher levels of FGF-2 when strained. 3) There is an increase in the sutural cell membrane permeability as a result of tensile strain. 4) The cells along the leading edges of the ossification fronts (at the insertion sites of Sharpeys fibers) demonstrated the maximum permeability increase. 5) There was an immediate (within seconds) increase in intracellular Ca ++. and 6) This increase in intracellular Ca ++ caused by tension was reversible and independent of the extracellular Ca ++ ion availability. In summary, these data support, in part, the conjecture that growth of the brain places strain on the cells within the immature sutures, which causes the iteration of a set of cellular subroutines. These subroutines integrate to generate the emergent property of directed cranial expansion with dissipation of the initiating strains.

Infections in craniofacial surgery: a combined report of 567 procedures from two centers.

&NA; This retrospective review of infectious complications was undertaken at two craniofacial centers (Dallas and Philadelphia). Fourteen infections were identified over a 6.5‐year period in 567 intracranial procedures primarily for craniosynostosis. There were no infections in infants under 13 months of age and no cases of meningitis. The overall infection rate was 2.5 percent, and 85 percent of infections occurred in secondary reoperative cases. Tracheostomies were not identified as a risk factor for infection. No difference was found in infection rates between patients with shaved and unshaved scalps. Candida and Pseudomonas were the two most common organisms identified, and 28 percent of our infections involved yeast. The average time to diagnose infection was 11.5 days (excluding three patients who averaged 5 months). Thirteen of the fourteen infections were treated surgically with placement of a subgaleal irrigation/drainage system. Initial bony debridement was kept to a minimum. Based on our findings, recommendations are made to further lower infection rates, particularly those caused by opportunistic organisms. (Plast. Reconstr. Surg. 100: 862, 1997.)

Critical review of microfixation in pediatric craniofacial surgery

The migration or passive intracranial translocation of microplates and screws in the pediatric craniofacial patient has been reported. A retrospective review was undertaken to clarify the incidence of microplate translocation and identify potential clinical implications. Computed tomographic imaging demonstrated internalization of microfixation in 14 of 27 pediatric patients. Statistically significant factors for microplate translocation include longer plates (p < 0.05) and those placed in the temporal region (p < 0.001). Younger patients and those with syndromic craniofacial dysostosis also had a higher incidence of translocation. Specific complications relating to the translocation of microplates were not found in any patient. The direct effects of translocated microplates and screws on the underlying brain and dura remain unclear.

Regional differences of dura osteoinduction: Squamous dura induces osteogenesis, sutural dura induces chondrogenesis and osteogenesis

&NA; Dura plays an important role in calvarial morphogenesis. However, precisely what that role is remains unclear. We present here in vivo evidence that dura without other central nervous system components induces both chondrogenesis and osteogenesis. The mechanism is, at least in part, by proximate tissue interaction. The objectives of this experiment were to answer the following: (1) Can dura actually induce osteogenesis without the influence of the underlying brain? (2) What are the requirements of this dura‐induced heterotopic osteogenesis? (3) What are the differences between dura underlying sutures and dura underlying the squamous portions of the cranial bones? Dura underlying the metopic, sagittal, and lambdoidal sutures and dura underlying the flat portions of frontal and parietal bones were obtained from neonatal Lewis rats and transplanted into the posterior thoraces of adult Lewis recipients. In group I, dura underlying the metopic, sagittal, and lambdoidal sutures (n = 20) and dura underlying the flat portions of frontal and parietal bones (n = 20) were transplanted individually into separate epitheliomesenchymal pockets. Group II animals had dura underlying the metopic, sagittal, and lambdoidal sutures (n = 10) and dura underlying the flat portions of frontal and parietal bones (n = 10) transplanted individually into surgically created mesenchymal pockets by placing the dura grafts between panniculus carnosus and latissimus dorsi muscles. The animals were sacrificed at 2‐week intervals. Light microscopy, special histochemical analysis, immunohistochemistry, and electron microscopy were performed. Bone formation was seen in 15 of the 18 animals (83 percent) in group I. No bone or cartilage formation was seen in group II. Chondrogenesis was seen in 4 animals receiving dura underlying the metopic, sagittal, and lambdoidal sutures in group I. Cellular hyperproliferation was seen at 2 weeks when dura was transplanted close to the hair follicles. These cells had a high nucleus‐to‐cytoplasm ratio and were positive for transforming growth factor beta. This hyperproliferation was followed by production and accumulation of Alcian bluepositive extracellular matrix that resisted digestion by hyaluronidase. Cellularly active cartilage was seen at 6 weeks. There was no chondrogenesis in animals receiving dura underlying the flat portions of frontal and parietal bones in group I. Electron microscopy demonstrated the presence of proteoglycan‐like ground substance and type II collagen in the inner layer of sutural dura and the predominance of dense type I collagen in the squamous dura and the external layer of the sutural dura. The important findings of this experiment are that (1) heterotopically transplanted neonatal dura can induce osteogenesis, (2) this heterotopic osteoinduction by dura requires epitheliomesenchymal interaction, and (3) separating dura into sutural dura and squamous dura, chondrogenesis occasionally occurred in addition to osteogenesis with the former, while only membranous ossification occurred with the latter, indicating intrinsic differences within the dura mater. This dural heterogeneity is supported by direct ultrastructural data. (Plast. Reconstr. Surg. 100: 23, 1997.)

Human PRRX1 and PRRX2 genes: Cloning, expression, genomic localization, and exclusion as disease genes for Nager syndrome

Abstract. In this study, we extend our examination of the function of the Prrx1 (a.k.a. Mhox, Prx1, K-2, and Pmx1) as well as Prrx2 (a.k.a. S8 and Prx2) genes by characterizing the expression of the human orthologs and their potential for causing specific human malformations. The expression pattern of PRRX2 and its close relative, PRRX1, were analyzed in human tissue by RT-PCR. Although the expression of these human genes is similar to their mouse orthologs, there are notable differences in expression. PRRX2 was detected in the human kidney and lung, whereas in mice and chickens neither of these tissues has been reported to express Prrx2. For PRRX1 the expression pattern was quite similar to other vertebrates, but the ratio of the two isoforms was reversed. To begin the search for the gene-disease connection, both genes were mapped to human chromosomes by FISH. The PRRX1 locus maps to 1q23, whereas the PRRX2 locus maps to 9q34.1. This localization, along with the recently described phenotypes of the gene-targeted Prrx1, Prrx2 and double mutant mice, enabled us to search the human disease databases for similar malformations. This examination suggested that mutations at the PRRX1 and/or PRRX2 loci could result in Nager Acrofacial Dysostosis (NAFD) syndrome. We obtained DNA samples from eight patients with NAFD, as well as two patients with Miller syndrome, and analyzed them for mutations in the PRRX1 and PRRX2 genes. The data excludes mutations in the presumed coding sequences of these genes from causing NAFD.

Effect of whole-body vibration on bone properties in aging mice

Recent studies suggest that whole-body vibration (WBV) can improve measures of bone health for certain clinical conditions and ages. In the elderly, there also is particular interest in assessing the ability of physical interventions such as WBV to improve coordination, strength, and movement speed, which help prevent falls and fractures and maintain ambulation for independent living. The current study evaluated the efficacy of WBV in an aging mouse model. Two levels of vibration--0.5 and 1.5g--were applied at 32Hz to CB57BL/6 male mice (n=9 each) beginning at age 18 months and continuing for 12 weeks, 30 min/day, in a novel pivoting vibration device. Previous reports indicate that bone parameters in these mice begin to decrease substantially at 18 months, equivalent to mid-fifties for humans. Micro-computed tomography (micro-CT) and biomechanical assessments were made in the femur, radius, and lumbar vertebra to determine the effect of these WBV magnitudes and durations in the aging model. Sera also were collected for analysis of bone formation and breakdown markers. Mineralizing surface and cell counts were determined histologically. Bone volume in four regions of the femur did not change significantly, but there was a consistent shift toward higher mean density in the bone density spectrum (BDS), with the two vibration levels producing similar results. This new parameter represents an integral of the conventional density histogram. The amount of high density bone statistically improved in the head, neck, and diaphysis. Biomechanically, there was a trend toward greater stiffness in the 1.5 g group (p=0.139 vs. controls in the radius), and no change in strength. In the lumbar spine, no differences were seen due to vibration. Both vibration groups significantly reduced pyridinoline crosslinks, a collagen breakdown marker. They also significantly increased dynamic mineralization, MS/BS. Furthermore, osteoclasts were most numerous in the 1.5 g group (p≤ 0.05). These findings suggest that some benefits of WBV found in previous studies of young and mature rodent models may extend to an aging population. Density parameters indicated 0.5 g was more effective than 1.5 g. Serological markers, by contrast, favored 1.5 g, while biomechanically and histologically the results were mixed. Although the purported anabolic effect of WBV on bone homeostasis may depend on location and the parameter of interest, this emerging therapy at a minimum does not appear to compromise bone health by the measures studied here.

A fractal analysis of human cranial sutures

OBJECTIVES Many biological structures are products of repeated iteration functions. As such, they demonstrate characteristic, scale-invariant features. Fractal analysis of these features elucidates the mechanism of their formation. The objectives of this project were to determine whether human cranial sutures demonstrate self-similarity and measure their exponents of similarity (fractal dimensions). DESIGN One hundred three documented human skulls from the Terry Collection of the Smithsonian Institution were used. Their sagittal sutures were digitized and the data converted to bitmap images for analysis using box-counting method of fractal software. RESULTS The log-log plots of the number of boxes containing the sutural pattern, N(r), and the size of the boxes, r, were all linear, indicating that human sagittal sutures possess scale-invariant features and thus are fractals. The linear portion of these log-log plots has limits because of the finite resolution used for data acquisition. The mean box dimension, D(b), was 1.29289 +/- 0.078457 with a 95% confidence interval of 1.27634 to 1.30944. CONCLUSIONS Human sagittal sutures are self-similar and have a fractal dimension of 1.29 by the box-counting method. The significance of these findings includes: sutural morphogenesis can be described as a repeated iteration function, and mathematical models can be constructed to produce self-similar curves with such D(b). This elucidates the mechanism of actual pattern formation. Whatever the mechanisms at the cellular and molecular levels, human sagittal suture follows the equation log N(r) = 1.29 log 1/r, where N(r) is the number of square boxes with sides r that are needed to contain the sutural pattern and r equals the length of the sides of the boxes.