Gli1+ Periodontal Mesenchymal Stem Cells in Periodontitis.

Periodontal mesenchymal stem cells (MSCs) play a crucial role in maintaining periodontium homeostasis and in tissue repair. However, little is known about how periodontal MSCs in vivo respond under periodontal disease conditions, posing a challenge for periodontium tissue regeneration. In this study, Gli1 was used as a periodontal MSC marker and combined with a Gli1-cre ERT2 mouse model for lineage tracing to investigate periodontal MSC fate in an induced periodontitis model. Our findings show significant changes in the number and contribution of Gli1+ MSCs within the inflamed periodontium. The number of Gli1+ MSCs that contributed to periodontal ligament homeostasis decreased in the periodontitis-induced teeth. While the proliferation of Gli1+ MSCs had no significant difference between the periodontitis and the control groups, more Gli1+ MSCs underwent apoptosis in diseased teeth. In addition, the number of Gli1+ MSCs for osteogenic differentiation decreased during the progression of periodontitis. Following tooth extraction, the contribution of Gli1+ MSCs to the tooth socket repair was significantly reduced in the periodontitis-induced teeth. Collectively, these findings indicate that the function of Gli1+ MSCs in periodontitis was compromised, including reduced contribution to periodontium homeostasis and impaired injury response.

Osteoblasts and MG-63 osteosarcoma cells behave differently when in contact with ProRoot MTA and White MTA.

AIM: To test the hypothesis that MG-63 osteosarcoma cells and primary osteoblasts react differently to ProRoot trade mark MTA (mineral trioxide aggregate) and White MTA by: (i) investigating the attachment of primary osteoblasts and MG-63 osteosarcoma cells to ProRoot trade mark MTA and White MTA; and (ii) comparing the osteogenic behaviour of both cell lines in contact with these endodontic materials. METHODOLOGY: Primary osteoblasts were harvested from foetal rat calvaria by sequential digestion and MG-63 osteosarcoma cells were purchased. Cells were exposed to ProRoot trade mark MTA and White MTA prepared according to the manufacturer's instructions. All samples and controls were prepared in quadruplicate. After 6, 9 and 13 days exposure to MTA, the cells were fixed and prepared for SEM examination. In addition, both the cell types were grown to confluence and exposed to beta-glycerophosphate and dexamethasone to assess mineralized nodule formation as a function of osteogenic behaviour. RESULTS: The number of cells on the surface of the culture dish and on top of the materials increased in all samples throughout the 3 time periods, except for White MTA where no primary osteoblasts were visible on top of the material by the end of 13 days. After exposing cells to differentiation medium nodules were observed in cultures of primary osteoblasts, but not of MG-63 osteosarcoma cells. CONCLUSIONS: Under the conditions of this study, whilst primary osteoblasts initially bound to White MTA, they did not survive on the surface by the end of 13 days. Primary osteoblasts formed mineralized nodules when exposed to differentiation medium, whilst MG-63 cells did not form nodules. As MG-63 cells do not behave osteogenically by forming mineralized nodules, and primary osteoblasts are more sensitive than MG-63 osteosarcoma cells to White MTA in cell culture, primary osteoblasts are more appropriate than MG-63 cells for testing endodontic materials in cell culture.

Correction of coronal suture synostosis using suture and dura mater allografts in rabbits with familial craniosynostosis.

OBJECTIVE: Resynostosis following surgical correction of craniosynostosis is a common clinical correlate. Recent studies suggest that the dura mater is necessary to maintain suture patency. It has also been hypothesized that dura mater from synostotic individuals may provide aberrant biochemical signals to the osteogenic fronts of the calvaria, which result in premature suture fusion and subsequent resynostosis following surgery. This study was designed to test this hypothesis by surgically manipulating the coronal suture and dura mater in rabbits with familial craniosynostosis to prevent postsurgical resynostosis. DESIGN: Craniofacial growth and histomorphometric data were collected from 129 rabbits: 72 normal controls and 57 rabbits with bilateral coronal suture synostosis (15 unoperated on controls; 13 surgical controls; 9 dura mater transplant only; 10 suture transplant only; and 10 suture and dura mater transplant). At 10 days of age, all rabbits had radiopaque amalgam markers placed on either side of the coronal, frontonasal, and anterior lambdoidal sutures. At 25 days of age, 42 synostosed rabbits had a 3 to 5-mm wide coronal suturectomy. Coronal sutures and/or underlying dura mater allografts were harvested from same-aged, wild-type, isohistogenic control rabbits and transplanted onto the dura mater of synostosed host rabbits. Serial radiographs were taken at 10, 25, 42, and 84 days of age, and the suturectomy sites were harvested at 84 days of age in 44 rabbits and serially sectioned for histomorphometric examination. RESULTS: Results revealed that cranial vault growth was significantly (p < .05) improved following surgical release of the fused coronal suture compared with synostosed rabbits who were not operated on but was still significantly different (p < .05) from that of normal control rabbits. By 84 days of age, significant (p < .05) differences were noted in calvarial suture marker separation, cranial vault shape indices, and cranial base angles between rabbits with and without dura mater allografts, probably as a result of resynostosis of the suturectomy site or suture-only allografts. Qualitative histological examination revealed that at 84 days of age rabbits with suture and dura allografts had patent coronal sutures, suture-only allografts had fused coronal sutures with extensive endosteal hyperostosis, dura mater-only allografts had some new bone in the suturectomy site that resembled rudimentary osteogenic fronts, and suturectomy controls had extensive endosteal bone formation and resynostosis of the suturectomy site. Significantly (p < .05) more bone was found in the suturectomy sites of rabbits without dura mater allografts compared with rabbits with dura mater allografts. CONCLUSIONS: Results support the initial hypothesis that normal dura mater allografts will maintain suture or suturectomy site patency and allow unrestricted craniofacial growth. However, it is still unclear whether the dura mater from normal rabbits was providing biochemical signals to the transplanted sutures or suturectomy sites or simply acting as a barrier to prevent abnormal biochemical signals from the dura mater of synostosed rabbits from reaching the calvaria. The clinical and therapeutic implications of these procedures are discussed.

A pilot study of short- and long-term sequelae to rigid fixation across metacarpal physes in a baboon model.

The use of rigid fixation for fractures of the extremities has become commonplace. The short- and long-term effects of rigid fixation on the growing hand, however, have not been studied thoroughly. In this project, the use of rigid fixation across metacarpal growth plates (physes) in growing primate hands was examined. The hypothesis to be tested was that long-term placement of rigid fixation devices across physes during stabilization of mid-shaft osteotomies will cause the physes to close prematurely. Fixation devices with screws placed in the epiphysis and left in place for 4 months or 1 year resulted in open physes, in support of the null hypothesis. However, in physes plated for 1 year, biochemical changes associated with increased bone differentiation were apparent. Findings suggest that rigid fixation across physes for as long as 1 year can be used appropriately in growing individuals when necessary. However, until additional investigation establishes whether the open physes are still capable of producing bone-lengthening hypertrophic chondrocytes, caution should be used in long-term placement of rigid fixation devices.

Elevated levels of transforming growth factors beta 2 and beta 3 in lambdoid sutures from children with persistent plagiocephaly.

OBJECTIVE: To analyze the pertinent history and physical findings specific to the subset of patients with a progressive posterior skull deformity, requiring surgery to correct their deformity. PATIENTS: Since the Academy of Pediatrics issued its recommendation on supine positioning of infants to prevent sudden infant death syndrome (SIDS) in 1992, 73 children have presented to the University of Virginia Craniofacial Anomalies Clinic with posterior-skull deformities. The majority were successfully managed with conservative therapy, but in six patients, the deformity was severe and persistent, requiring surgical correction. All six children were older (7.5-12 mo), presenting with more severe morphologic appearances and a higher incidence of associated neurodevelopmental delay. Three had family backgrounds of isolated craniosynostosis. METHODS: Characteristics of these patients were examined to determine why they may have differed from those that responded to conservative management. Immunohistochemical staining of their lambdoid sutures was performed. RESULTS: Significantly increased staining for TGF-beta 2 and TGF-beta 3, potent stimulators of bone cell growth and differentiation, was seen in all 'affected' sutures from the flattened side of the skull, compared to unaffected sutures from the protruding side of the skull-a pattern similar to that seen during normal bony obliteration of calvarial sutures. CONCLUSION: The majority of patients with posterior plagiocephaly associated with positioning responded to conservative management, while a small subset of patients with persistent posterior skull deformation required surgical intervention. A genetic basis for the latter patients' persistent plagiocephaly, rather than positioning, cannot be ruled out. Genetics, prolonged external pressure against the sutures, or a combination of these factors may lead to permanently raised levels of growth factors in 'affected' sutures.

Repair of critical size rat calvarial defects using extracellular matrix protein gels.

In this study the authors examined the capacity of gels of reconstituted basement membrane, laminin, and type I collagen to mediate repair of critical size defects in rat calvaria. Although autografts are widely used to repair bone defects caused by trauma or surgical treatment of congenital malformations, neoplasms, and infections, an adequate quantity of graft is not always available. Allogenic bone is readily available, but its use is associated with an increased incidence of nonunion, fatigue fracture, and rejection. Biologically active, purified components of basement membranes, which have been shown to promote osteogenic differentiation and angiogenesis in vitro and type I collagen (the major constituent of bone extracellular matrix) can be formed into native isotonic space-filling gels. In this study critical size calvarial defects were created in retired male Sprague-Dawley rats. Thirty-six animals were divided into seven groups. Group 1 (control) received no treatment for the defects. Group 2 animals were implanted with methylcellulose. Groups 3, 4, 5, and 6 were implanted with gels of type I collagen, reconstituted basement membrane, or laminin, respectively. The last group of three animals (Group 7) was implanted with 100 micrograms of type I collagen gels (identical to Group 3) and sacrificed at 20 weeks following a single CT scan to determine if complete healing could be obtained with this method given sufficient time. Except for rats in the type I collagen group that was evaluated by multiple computerized tomography (CT) scans biweekly from 2 to 12 weeks, bone repair was evaluated using CT at 12 weeks. Healing was quantified using three-dimensional reconstruction of CT. Following the final CT scan in each experimental group, animals were sacrificed, and a sample of tissues was evaluated by conventional histology. Animals treated with type I collagen gels showed 87.5% repair of the area of the defects at 12 weeks and 92.5% repair by 20 weeks. Increasing the gel volume 1.5 x accelerated complete repair to 3 months. Murine-reconstituted basement membrane and laminin gels induced 55.5% and 46.3% repair, respectively, at 3 months. In untreated control animals 7% repair of the area of the defects showed at 3 months. Histological analysis confirmed new bone formation in partial and completely healed defects. Bioengineered native collagen gels may have wide applicability for bone repair as an alternative bone graft material alone, in combination with autograft or marrow aspirate, or as a delivery system for osteogenic growth factors.

In the absence of periosteum, transplanted fetal and neonatal rat coronal sutures resist osseous obliteration.

Normal craniofacial development depends on expansion of the cranial vault by growth at the sutures. Inappropriate development of the sutures leads to global disruption of patterns of craniofacial growth. Tissue interactions between dura mater and suture matrix play a critical role in the phenotypic maintenance of cranial sutures. However, the function of the periosteum in this process remains under-reported and controversial. To examine the contribution of periosteum in maintaining the patency of coronal sutures, fetal and neonatal rat coronal sutures were transplanted to surgically created defects in adult rat host parietal bones. These sutures were examined for their ability to persist in the host milieu in the presence and absence of both donor and host periosteum. This study established that removal of both host and transplant periosteum, unlike removal of dura mater, did not lead to obliteration of either fetal or neonatal sutures. Thus, periosteum and dura mater are nonequivalent tissues with respect to influence on suture patency.

Estrogen inhibits calbindin-D28k expression in mouse uterus.

The cellular localization and hormonal controls of calbindin-D9k expression in the rodent reproductive tract have suggested new functions for this protein. The present studies were undertaken to extend the earlier studies of calbindin-D9k to the related protein, calbindin-D28k. Immunohistochemical studies revealed that calbindin-D28k was absent from female rat reproductive tissues, but was abundantly expressed in immature mouse uterus and oviduct. Immunoreactivity was restricted to the endometrial and glandular epithelium of the uterus and the oviductal epithelium. Neither 1,25-dihydroxyvitamin D- nor strontium-containing diets (to blunt 1,25-dihydroxyvitamin D production) affected expression of calbindin-D28k. Uterine, but not oviductal, calbindin-D28k decreased markedly at sexual maturity; this pattern persisted in pregnant mice and was reproduced in immature mice by the administration of estradiol (3 micrograms/day for 3 days). RNA extraction and Northern analyses demonstrated that estrogen markedly decreased calbindin-D28k mRNA abundance in the uterus, but not in the oviduct. These findings suggest that estrogen affects mammalian calbindin-D28k expression and represent a rare example of estrogen-induced down-regulation of gene expression.