Biomedical subjects

M T Longaker

Publications and source records attributed to M T Longaker.

At least 91 records · Page 5Linked to original sources

PubMed2000

"Pumping the regenerate": an evaluation of oscillating distraction osteogenesis in the rodent mandible.

Mandibular distraction osteogenesis (DO) has become an important technique to lengthen the hypoplastic mandible and to reconstruct osseous defects after ablative surgery. The hallmark of successful DO is the creation of new bone within the distraction gap. Several anecdotal reports have described alternating compressing and lengthening protocols (i.e., "pumping the regenerate") to augment regenerate bone formation. The purpose of this experiment was to analyze formally the effects of an alternating compression/distraction protocol with a traditional distraction protocol. Ten adult male rats underwent unilateral mandibular osteotomy with placement of a custom distractor. After a latency period of 5 days, distraction was initiated at a rate of 0.25 mm twice daily. Animals in the control group (N = 5) were distracted to a length of 5.0 mm for 10 days at a rate of 0.25 mm twice daily. In contrast, animals in the experimental group (N = 5) were distracted to a length of 2.5 mm (at a rate of 0.25 mm twice daily) for 5 days, then compressed 1.0 mm for a 2-day period, and redistracted to a length of 5.0 mm. Regenerate cross-sectional area was evaluated by computed tomography performed after 5 weeks of consolidation. Gross examination and histological analysis were performed by a panel of experienced reviewers. Radiological as well as histological analysis of regenerate cross-sectional area demonstrated no significant differences between experimental (i.e., "pumped") and control groups. Both groups demonstrated excellent regenerate bone formation with no evidence of fibrous union. This study represents the first attempt to investigate the anecdotal technique of pumping the mandibular regenerate. The authors have demonstrated that pumping the regenerate leads to no substantial differences in radiological or histological appearance of regenerate bone formation.

Animals↗

PubMed2000

Immature versus mature dura mater: II. Differential expression of genes important to calvarial reossification.

The ability of immature animals and newborns to orchestrate successful calvarial reossification is well described. This capacity is markedly attenuated in mature animals and in humans greater than 2 years of age. Previous studies have implicated the dura mater as critical to successful calvarial reossification. The authors have previously reported that immature, but not mature, dural tissues are capable of elaborating a high expression of osteogenic growth factors and extracellular matrix molecules. These findings led to the hypothesis that a differential expression of osteogenic growth factors and extracellular matrix molecules by immature and mature dural tissues may be responsible for the clinically observed phenotypes (i.e., immature animals reossify calvarial defects; mature animals do not). This study continues to explore the hypothesis through an analysis of transforming growth factor (TGF)-beta3, collagen type III, and alkaline phosphatase mRNA expression. Northern blot analysis of total RNA isolated from freshly harvested immature (n = 60) and mature (n = 10) dural tissues demonstrated a greater than three-fold, 18-fold, and nine-fold increase in TGF-beta3, collagen type III, and alkaline phosphatase mRNA expression, respectively, in immature dural tissues as compared with mature dural tissues. Additionally, dural cell cultures derived from immature (n = 60) and mature dura mater (n = 10) were stained for alkaline phosphatase activity to identify the presence of osteoblast-like cells. Alkaline phosphatase staining of immature dural cells revealed a significant increase in the number of alkaline phosphatase-positive cells as compared with mature dural tissues (p < 0.001). In addition to providing osteogenic humoral factors (i.e., growth factors and extracellular matrix molecules), this finding suggests that immature, but not mature, dura mater may provide cellular elements (i.e., osteoblasts) that augment successful calvarial reossification. These studies support the hypothesis that elaboration of osteogenic growth factors (i.e., TGF-beta33) and extracellular matrix molecules (i.e., collagen type III and alkaline phosphatase) by immature, but not mature, dural tissues may be critical for successful calvarial reossification. In addition, these studies suggest for the first time that immature dural tissues may provide cellular elements (i.e., osteoblasts) to augment this process.

Aging↗

PubMed2000

A molecular analysis of the isolated rat posterior frontal and sagittal sutures: differences in gene expression.

Although it is one of the most commonly occurring craniofacial congenital disabilities, craniosynostosis (the premature fusion of cranial sutures) is nearly impossible to prevent because the molecular mechanisms that regulate the process of cranial suture fusion remain largely unknown. Recent studies have implicated the dura mater in determining the fate of the overlying cranial suture; however, the molecular biology within the suture itself has not been sufficiently investigated. In the murine model of cranial suture fusion, the posterior frontal suture is programmed to begin fusing by postnatal day 12 in rats (day 25 in mice), reliably completing bony union by postnatal day 22 (day 45 in mice). In contrast, the sagittal suture remains patent throughout the life of the animal. Using this model, this study sought to examine for the first time what differences in gene expression--if any--exist between the two sutures with opposite fates. For each series of experiments, 35 to 40 posterior frontal and sagittal suture complexes were isolated from 6-day-old Sprague-Dawley rat pups. Suture-derived cell cultures were established, and ribonuicleic acid was derived from snap-frozen, isolated suture tissue. Results demonstrated that molecular differences between the posterior frontal and sagittal suture complexes were readily identified in vivo, although these distinctions were lost once the cells comprising the suture complex were cultured in vitro. Hypothetically, this change in gene expression resulted from the loss of the influence of the underlying dura mater. Significant differences in the expression of genes encoding extracellular matrix proteins existed in vivo between the posterior frontal and sagittal sutures. However, the production of the critical, regulatory cytokine transforming growth factor beta-1 was equal between the two suture complexes, lending further support to the hypothesis that dura mater regulates the fate of the overlying cranial suture.

Animals↗

PubMed2000

Response to tissue injury.

Cutaneous injury, whether by laser, chemical, or scalpel, results in scar formation. The normal response to such an insult occurs in the middle of a continuum of wound repair processes. On one end of the continuum are the overhealed responses (i.e., keloids). On the opposite end are tissue regeneration and scarless healing as seen in fetal wounds. This article reviews the molecular biology and mechanisms leading to these various clinical phenotypes and discusses future potential modalities that may replicate the scarless wound healing mechanism in adults.

Adult↗

PubMed1999

Cutaneous rat wounds express c49a, a novel gene with homology to the human melanoma differentiation associated gene, mda-7.

We have used DD-PCR (differential display-polymerase chain reaction) to identify new genes that are over- or underexpressed during wound repair. DD-PCR performed on excisional wounds identified the expression of rat c49a. Cloning and sequence analysis of the rat c49a gene revealed high homology to a novel human melanoma differentiation associated gene, mda-7. The human mda-7gene isolated from melanoma cell lines, has been linked with human melanoma differentiation, and growth suppression. Moreover, transfection of human mda-7 constructs into human tumor cells suppresses the growth and colony formation of tumor cells from diverse origins. To confirm and relatively quantitate expression of rat c49a gene during repair, specific primer, reduced cycle RT-PCR (reverse transcription-PCR) was performed. RT-PCR showed an approximately 9 to 12-fold elevation of rat c49a mRNA at 12 h to 5 days above nonwounded controls that gradually decreased to approximately 1.5 to 3-fold by day 14. Cloning and sequence analysis of the entire 1200 base pair c49a gene product showed 78% nucleotide homology to human mda-7. Immunohistochemistry studies localized rat C49A expression primarily to fibroblast-like cells at the wound edge and base. The marked up-regulation of rat c49a transcripts during the inflammatory and early granulation tissue phases of wound repair where cellular processes such as re-epithelialization, angiogenesis, and fibroplasia predominate--suggest that c49a is associated with proliferation of fibroblasts in wound healing.

Amino Acid Sequence↗

PubMed1999

Fibroblast response to hypoxia: the relationship between angiogenesis and matrix regulation.

A number of studies have demonstrated the critical role of angiogenesis for successful wound repair in the surgical patient. Vascular disruption from tissue injury due to trauma or surgery leads to a hypoxic zone in the healing wound. In this dynamic process, angiogenesis is vital for the delivery of oxygen, nutrients, and growth factors necessary to initiate the synthetic processes of wound healing. Fibroblasts, invading the wound early in the healing process, are involved in extracellular matrix (ECM) deposition as well as wound contraction. However, the exact mechanisms by which important genes are regulated remain unknown. In order to examine these processes, we studied the effects of hypoxia on fibroblasts for the expression of VEGF, type IalphaI collagen, and matrix-metalloproteinase-3, three genes essential for the regulation of angiogenesis, ECM deposition, and ECM degradation in wound healing. Primary cell cultures of normal human dermal fibroblasts (NHDFs) were placed in hypoxia for varying periods of time. Northern blot hybridization was performed with [alpha32P]dCTP-labeled cDNA probes for VEGF, type IalphaI collagen, and MMP-3. The results demonstrated a time-dependent VEGF mRNA upregulation (470% of baseline) under hypoxia. Type IalphaI collagen increased (170% of baseline) at 24 h, but was then abruptly downregulated to 3.8% of baseline at 48 h. MMP-3 was incrementally downregulated to 2.2% of baseline at 48 h. These experiments focused on the effect of hypoxia on genes thought to play a role in wound repair. VEGF upregulation in the hypoxic microenvironment of the early wound may serve to stimulate angiogenesis. Type IalphaI collagen, though upregulated early on, was abruptly downregulated at 48 h. This downregulation may reflect the in vivo requirement for angiogenesis to deliver oxygen for successful hydroxylation and collagen synthesis in the wound. MMP-3, also downregulated at 48 h, may also implicate the need for angiogenesis. These data support the theory that hypoxia-driven angiogenesis is critical for ECM formation and remodeling in successful soft tissue repair. Furthermore, they may represent the role of hypoxia as an important regulator to efficiently balance these complex processes in the healing wound.

Cell Hypoxia↗

PubMed1999

Downregulation of apoptosis-related genes in keloid tissues.

BACKGROUND: Physiologically programmed cell death or apoptosis occurs during the natural balance between cellular proliferation and demise. MATERIALS AND METHODS: We compared the expression of 64 apoptosis-related genes in keloids and normal scars to investigate the potential role of apoptosis in keloid formation. Two sets of mRNA were isolated from keloids excised from four previously untreated patients and four normal scar patients separately. Human cDNA arrayed hybridization was performed to compare the apoptosis-related gene expression between these two groups. In addition, TUNEL assays were performed to evaluate the percentage of apoptotic cells in keloids (center and periphery) versus normal scars. RESULTS: Eight of the sixty-four apoptosis-related genes studied were significantly underexpressed in keloid tissue. The underexpressed genes and their relative expression compared with normal scar were defender against cell death 1 (DAD-1) (34.1% of normal scar); nucleoside diphosphate kinase B (c-myc transcription factor) (24.7%); glutathione S-transferase (17.9%); glutathione S-transferase microsomal (28.1%); glutathione peroxidase (47.2%); tumor necrosis factor receptor 1-associated protein (TRADD) (51.0%); 19-kDa interacting protein 3 (NIP3) (36.0%); and cytoplasmic dynein light chain 1 (HDLC1) (47.7%). Spatial analysis of apoptosis using TUNEL assays revealed apoptosis indices of 0.83 for keloid periphery and 0.63 for keloid center. CONCLUSIONS: In this study we demonstrated underexpression of apoptosis-related genes in human keloid tissue and decreased apoptotic activity in fibroblasts derived from keloids versus normal scars. We hypothesized that keloid fibroblasts fail to undergo physiologically programmed cell death and, thus, continue to produce and secrete connective tissue beyond the period expected in normal scar formation, accounting for the progressive and hypertrophic nature of keloids. This mechanism leads to new possibilities for treatment of keloids through induction of apoptosis.

Adolescent↗

PubMed1999

TTF-1 and HNF-3beta in the developing tracheoesophageal fistula: further evidence for the respiratory origin of the distal esophagus'.

PURPOSE: Using an established rat model of esophageal atresia with tracheoesophageal fistula (EA-TEF), the authors have studied the organogenesis of this congenital anomaly. The authors previously have proposed that the "distal esophagus" actually is of respiratory lineage. In this report this hypothesis is tested by examining the expression of two foregut patterning transcription factors, thyroid transcription factor-1 (TTF-1) and hepatocyte nuclear factor-3beta (HNF-3beta), within the developing TEF. METHODS: Pregnant Sprague-Dawley rats were injected with 2.2 mg/kg of Adriamycin intraperitoneally on days 6 to 9 of gestation. Using microdissection, the trachea, blind-ending esophagus, TEF, and stomach were isolated from embryos of various gestional ages. Immunohistochemistry was performed using polyclonal antibodies to TTF-1 and HNF-3beta. RESULTS: TTF-1 is a homeodomain protein that previously has been shown to be expressed in the lung and trachea but not in the gastrointestinal tract, and which, when deleted in a developing lung, results in a mouse with no peripheral lung parenchyma. TTF-1 was expressed strongly in the lung, fistula, and distal esophagus, but not in the proximal esophagus. HNF-3beta is a forkhead transcription factor important in foregut patterning that binds and activates the TTF-1 promotor sequence. HNF-3beta was expressed globally in the fistula and lung as well as the esophagus. CONCLUSIONS: The expression of the lung-specific transcription factor TTF-1 within the TEF strongly implies that the "distal esophagus" is a respiratory-derived structure and thus supports our theory of TEF organogenesis. The conservation of HNF-3beta expression both in the TEF as well as the normal developing trachea and esophagus suggests that global foregut patterning is intact in the formation of this anomaly, and the defect lies at the level of the respiratory versus gastrointestinal commitment.

Animals↗

PubMed1999

Esophageal atresia with tracheoesophageal fistula: suggested mechanism in faulty organogenesis.

BACKGROUND/PURPOSE: The organogenesis of esophageal atresia with tracheoesophageal fistula (EA-TEF) is unknown. Using an established model for EA-TEF in rats, the authors proposed to study this aberrancy of development in the hope of gaining insight into its mechanism of formation. METHODS: Pregnant Sprague-Dawley rats were injected with 2.2 mg/kg of Adriamycin intraperitoneally on days 6 through 9 of gestation. Using microdissection, the trachea, blind-ending esophagus, TEF, and stomach were isolated from embryos of various gestional ages. The specimens were analyzed histologically with routine H&E staining. RESULTS: The classic EA-TEF developed in the embryos, with proximal EA and distal TEF. As expected, the atresia formed as a blind-ending pouch, but the distal fistula began as an apparent equal trifurcation of the tracheal anlage into two mainstem bronchi and the fistula tract leading to the stomach. Histological analysis of the fistula tract showed respiratorylike pseudostratified columnar epithelium. CONCLUSIONS: TEF develops as the middle branch of a tracheal trifurcation. EA-TEF occurs by a primary atresia of the esophagus. As a secondary phenomenon, the distal foregut anlage is switched toward the pulmonary phenotype. It trifurcates, and its middle branch grows caudally to fistulize into the stomach.

Animals↗

PubMed1999

The ontogeny of TGF-beta1, -beta2, -beta3, and TGF-beta receptor-II expression in the pancreas: implications for regulation of growth and differentiation.

BACKGROUND/PURPOSE: The transforming growth factor-beta (TGF-beta) cytokines are important regulators of growth and differentiation in multiple mammalian organ systems. Recent studies suggest that they may play a significant role in the regulation of pancreatic organogenesis. The authors proposed to examine the ontogeny of expression of the TGF-beta cytokine isoforms (TGF-beta1, beta2, and beta3), as well as that of the type II TGF-beta receptor (TbetaRII), in the pancreas. We hypothesized that their patterns of expression might help to clarify the manner in which they influence the development of this organ. METHODS: Embryos from pregnant CD-1 mice were harvested on gestational days 12.5, 15.5, and 18.5. Microdissection was performed on the embryos to isolate their pancreases. The pancreases were fixed, frozen embedded, and sectioned with a cryostat. Immunohistochemistrywas performed using polyclonal antibodies to TGF-beta1, beta2, and beta3, and TbetaRII. RESULTS: The patterns of expression of TGF-beta1, beta2, and beta3 were similar throughout gestation. They were all present, though weakly, early in the development of the pancreas, in the E12.5 epithelial cells. Their expression persisted and became localized to the acinar cells later in gestation. TbetaRII staining was present in both the E12.5 epithelial cells and the surrounding mesenchyme. As the pancreas developed, TbetaRII became strongly expressed in the ductal epithelial cells with only minimal staining in the acinar and endocrine cells. CONCLUSIONS: TGF-betas may play a role in regulating pancreatic organogenesis. Our data suggest that they may be required for the normal development of acini. As in other cell systems, TGF-beta1 may act as a suppressor of pancreatic cellular growth and differentiation. The localization of TbetaRII to the mature ductal epithelium may indicate a need for ongoing regulation of growth and differentiation in the pancreatic ducts beyond the fetal period.

Animals↗

PubMed1999

Epithelio-mesenchymal interactions in the developing mouse pancreas: morphogenesis of the adult architecture.

BACKGROUND/PURPOSE: The mammalian pancreas is thought to develop through a complex interaction between the budding epithelium and the surrounding mesenchyme. The exact nature of this interaction is unclear. Most of what is known to date of these interactions comes from a series of organ culture experiments done in the late 1960s. Nevertheless, these important experiments may have been confounded by less-defined culture media and organ dissection techniques, because the results are not reproducible in our hands. The authors undertook a study to reexplore these basic epithelio-mesenchymal interactions. METHODS: Using previously described organ dissection and culture techniques the authors examined the basic interactions between the embryonic pancreatic epithelium and its mesenchyme with histological and immunohistological techniques. RESULTS: The authors found that, contrary to previous reports, the earliest pancreatic anlage did not possess the intrinsic signaling necessary to support normal growth and differentiation in vitro. Intimate contact between the epithelium and the mesenchyme may be necessary until E11.5 for normal growth and differentiation. The age of the mesenchyme seemed to correlate with the degree of acinar differentiation, and proximity of mesenchyme was important for acinar differentiation. CONCLUSIONS: Previous investigations into the basic epithelio-mesenchymal interactions in the developing mammalian pancreas may have had confounding factors. Extrinsic signals seem necessary for complete pancreatic differentiation, and mesenchymal factors appear important for acinar differentiation.

Animals↗

PubMed1999

Vascular development in the mouse embryonic pancreas and lung.

PURPOSE: The purpose of this study was to analyze the formation of blood vessels in the developing mouse pancreas and lung by studying two ligands, angiopoietin-1 (ang1) and angiopoietin-2 (ang2), which are thought to play a role as angiogenesis-activating factors in development. Understanding the role of vasculogenic peptides in normal embryonic development also may have important implications for common clinical problems regarding neonatal pulmonary vasculature. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) as well as Southern blotting was used to determine the ontogeny of angiopoietin-1 and angiopoietin-2 gene expression in the embryonic mouse pancreas and lung. Immunohistochemistry was performed for von Willebrand factor, a known marker of endothelial cells, to chronicle the development of the vasculature in these organs. RESULTS: The authors determined the temporal expression of angiopoietin-1 and angiopoietin-2 as a function of gestational age. RT-PCR data demonstrated expression of ang1 and ang2 in the developing mouse lung between gestational day E9.5 and postnatal day 1, and in the developing pancreas between gestational days E12.5 and E18.5. Southern blot analysis confirmed PCR data for ang2 expression in both the lung and pancreas. The authors also traced the spatial development of the vascular system by von Willebrand factor staining. For both lung and pancreas specimens, no blood vessels were identifiable by immunohistochemistry until embryonic day 12.5. With increased gestational age, the blood vessel networks grew larger. CONCLUSION: The authors have demonstrated that ang1 and ang2 may be involved in the mechanisms of vascular development in the embryonic mouse lung and pancreas.

Angiopoietin-1↗

PubMed1999

Molecular approaches to understanding organogenesis.

The elucidation of the molecular mechanisms of mammalian organogenesis is the foundation on which we can build an improved understanding of organ pathology and pathophysiology. This paper uses the lung and the pancreas as paradigms to demonstrate how advances in basic molecular developmental biology research has translated into new appreciation of, and even novel potential treatment strategies for, congenital anomalies and mature diseases of these organs.

Animals↗

PubMed1999

Fetal wound repair: where do we go from here?

In contrast to adult wound healing, early-gestation fetal skin wound healing occurs rapidly, in a regenerative fashion, and without scar formation. The accelerated rate of healing, relative lack of an acute inflammatory response, and an absence of neovascularization distinguishes fetal from adult wound healing. However, this remarkable ability of the fetus to heal without scarring still remains poorly understood. The uncertainties include the role of cytokines, extracellular matrix components, homeobox genes, and certain cell types in the scarless wound repair process. Nevertheless, some strides have been made within the last two decades. This report, discusses the current knowledge of the mechanisms and characteristics of scarless fetal wound healing. Furthermore, to shy away from being just another all inclusive review, the authors point out deficiencies in the knowledge base on this important topic. Last, the future direction of research is discussed that may elucidate the mechanisms regulating the scarless repair phenomena.

Cicatrix↗

PubMed1999

Techniques for applying subatmospheric pressure dressing to wounds in difficult regions of anatomy.

Subatmospheric pressure dressing (SPD) has been commercially available in the United States since 1995 as the vacuum-assisted closure (VAC) device. SPD increases local blood flow, decreases edema and bacterial count, and promotes the formation of granulation tissue. Despite recent clinical successes with the use of SPD in a variety of wound types, problems may occur with application of VAC system in certain areas of the body. The main limitation occurs when attempting to maintain an airtight seal over irregular surfaces surrounding a wound. For example, application of the adhesive drape and creation of a seal are particularly difficulty in the hip and perineum. In addition, wounds of the lower extremity can occur in multiple sites, posing the problem of providing a vacuum dressing to more than one wound from one suction pump machine. To address these challenging clinical wounds, we have developed techniques to allow the successful application of SPD to sacral pressure ulcers near the anus, and to multiple large lower extremity ulcers.

Adhesives↗

PubMed1999

Angiogenesis during mandibular distraction osteogenesis.

Recruitment of a blood supply is critical for successful bone induction and fracture healing. Despite the clinical success of distraction osteogenesis (DO), an analysis of angiogenesis during membranous bone DO has not been performed. The purpose of this study was to evaluate the temporal and spatial pattern of angiogenesis during mandibular DO. The right hemimandible of adult male rats was osteotomized, and a customized distraction device was applied. Following a 3-day latency period, distraction was begun at a rate of 0.25 mm twice daily for 6 days (3.0 mm total; 12% increase in mandibular length). Three animals each were sacrificed on days 2, 4, and 6 of distraction (D1, D2, and D3 respectively), or after 1, 2, or 4 weeks of consolidation (C1, C2, and C3 respectively). Two experienced pathologists reviewed the regenerate histology, and angiogenesis was assessed by counting the number of blood vessels per intermediate-power field (IPF). Statistical analysis was performed using analysis of variance, with p < or = 0.05 considered significant. Results demonstrate that mandibular DO was associated with an intense vascular response during the early stages of distraction (D1). On average, 31.5+/-7.9 vessels were noted in each IPF examined during this time point. The number of blood vessels in the distraction regenerate decreased significantly during the later distraction time points, with approximately 14.0+/-2.0 and 14.7+/-3.5 blood vessels per IPF in sections obtained after days 4 and 6 of distraction (D2, D3) respectively. However, blood vessels at these time points took on a more mature histological pattern. During the consolidation period, the number of blood vessels noted in the regenerate decreased with 8.0+/-2.6, 9.3+/-2.1, and 4.0+/-2.0 vessels per IPF in sections obtained after 1, 2, or 4 weeks of consolidation (C1, C2, C3) respectively (p < 0.05 compared with vessel counts during the earliest distraction time point). This study demonstrates for the first time that an intense vascular response associated with mandibular DO occurs primarily during the early stages of distraction. The authors hypothesize that as distraction continues, newly formed vessels likely undergo consolidation, thus forming more mature vessels capable of withstanding distraction forces. Future studies will assess the effects of therapeutic interventions designed to increase angiogenesis during DO on bony regenerate formation.

Animals↗

PubMed1999

Gene expression of insulin-like growth factors I and II in rat membranous osteotomy healing.

Poorly healing mandibular osteotomies can be a difficult problem in reconstructive surgery. Many therapies have been attempted to augment the healing of mandibular fractures, defects, or osteotomies, but these methods have substantial drawbacks or have been ineffective. The difficulty in treating poorly healing bony defects has led to the exploration of gene therapy as a possible approach to supplement or accelerate mandibular fracture healing. To understand at what point the introduction of a suitable gene candidate might be of benefit in mandibular healing, it is imperative to examine the temporal expression of bone growth factors in a model of membranous bone healing. Insulinlike growth factors (IGFs) I and II are two such bone growth factor candidates because of their known potent in vitro as well as in vivo effects on bone formation. In this study the authors demonstrate the temporal pattern of IGF I and IGF II gene expression during mandibular osteotomy healing using a rat model. Their data reveal that IGF I and IGF II were elevated 7 days after a mandibular osteotomy that was held in external fixation. The upregulation of IGF I and IGF II during mandibular bone healing underscores the importance of these growth factors in bone repair. Gene therapy utilizing recombinant viral constructs containing IGFs I and II may be of benefit during mandibular bone healing in an effort to augment clinical scenarios of poor or retarded bony repair.

Animals↗

PubMed1999

Analysis of TGF-beta production by fusing and nonfusing mouse cranial sutures in vitro.

The role of transforming growth factor beta (TGF-beta) in the regulation of cranial suture fusion has been studied by various qualitative techniques such as in situ hybridization and immunohistochemistry. Although the relative expression of TGF-beta isoforms has been assessed in these studies, increased expression of TGF-beta has not been demonstrated in a quantitative fashion. Therefore, the purpose of this study was to quantify TGF-beta production by fusing (posterofrontal [PF]) and nonfusing (sagittal) mouse sutures using two different quantitative TGF-beta assays. The PF and sagittal sutures of 25-day-old mice were harvested and cultured separately in vitro. Culture media conditioned for 48 hours were collected after 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 days of culture, and total TGF-beta production was assessed using a TGF-beta bioassay. For a quantitative TGF-beta1 immunoassay, media conditioned for 48 hours were collected after 3, 5, 7, 9, 14, 22, and 28 days of culture. The TGF-beta bioassay revealed large amounts of total TGF-beta activity in both PF and sagittal sutures during the first week of culture, with decreasing amounts thereafter. Absolute TGF-beta activity in conditioned media collected from PF sutures at several early time points was higher than those obtained from sagittal sutures; however, these differences were not statistically significant. The results of the TGF-beta1 immunoassay (enzyme-linked immunosorbent assay) were similar to the bioassay in that the highest TGF-beta1 levels were noted during the first week of culture period and decreased thereafter. Analysis of variance of these samples, however, revealed significantly more TGF-beta1 protein in samples collected from the PF suture compared with the sagittal suture on days 3 and 5 of culture (p < 0.05). TGF-beta1 levels in the conditioned media obtained from PF sutures remained elevated compared with the sagittal suture on days 7 and 9; however, these differences were not statistically significant. Increased production of TGF-beta in the conditioned media of fusing PF sutures is the first such quantitative demonstration of growth factor upregulation during suture fusion and supports the hypothesis that TGF-beta expression may be important in cranial suture fusion.

Animals↗