Biomedical subjects

Kang Ting

Publications and source records attributed to Kang Ting.

18 recordsLinked to original sources

PubMed2007

Craniosynostosis-associated gene nell-1 is regulated by runx2.

UNLABELLED: We studied the transcriptional regulation of NELL-1, a craniosynostosis-related gene. We identitifed three OSE2 elements in the NELL-1 promoter that are directly bound and transactivated by Runx2. Forced expression of Runx2 induces NELL-1 expression in rat calvarial cells. INTRODUCTION: We previously reported the upregulation of NELL-1 in human craniosynostosis and the overexpression of Nell-1 in transgenic animals that induced premature suture closure associated with increased osteoblast differentiation. To study the transcriptional regulation of NELL-1, we analyzed the 5' flanking region of the human NELL-1 gene. We identified three osteoblast specific binding elements 2 (OSE2) sites (A, B, and C) within 2.2 kb upstream of the transcription start site and further studied the functionality of these sites. MATERIALS AND METHODS: An area of 2.2 kb and a truncated 325 bp, which lacked the three OSE sites, were cloned into a luciferase reporter gene, and co-transfected with Runx2 expression plasmid. The three OSE2 sites were individually mutated and co-transfected with Runx2 expression plasmid into Saos2 cells. Gel shifts and supershifts with Runx2 antibodies were used to determine specific binding to OSE2 sites. CHIP assays were used to study in vivo binding of Runx2 to the Nell-1 promoter. Runx2 expression plasmid was transfected into wildtype and Runx2(-/-) calvarial cells. Nell-1, osteocalcin, and Runx2 expression levels were measured using RT-PCR. RESULTS: Addition of Runx2 dose-dependently increased the luciferase activity in the human NELL-1 promoter-luciferase p2213. The p325 truncated NELL-1 construct showed significantly lower basal level of activity. Nuclear extract from Saos2 cells formed complexes with site A, B, and C probes and were supershifted with Runx2 antibody. Mutation of sites A, B, and C significantly decreased basal promoter activity. Furthermore, mutation of sites B and C had a blunted response to Runx2, whereas mutation of site A had a lesser effect. Runx2 bound to NELL-1 promoter in vivo. Transfection of Runx2 in rat osteoblasts upregulated Nell-1 and Ocn expression, and in Runx2 null calvarial cells, both Nell-1 and Ocn expression were rescued. CONCLUSIONS: Runx2 directly binds to the OSE2 elements and transactivates the human NELL-1 promoter. These results suggest that Nell-1 is likely a downstream target of Runx2. These findings may also extend our understanding of the molecular mechanisms governing the pathogenesis of craniosynostosis.

Animals↗

PubMed2006

The osteoinductive properties of Nell-1 in a rat spinal fusion model.

BACKGROUND CONTEXT: Recombinant growth factors bone morphogenetic protein-2 (BMP-2) and BMP-7 are currently approved for human use but are associated with various adverse effects including ectopic bone formation and local inflammatory reaction. The development of alternative growth factors may help minimize the adverse effects of current osteoinductive therapeutics. Nell-1 (Nel-like molecule-1; Nel [a protein strongly expressed in neural tissue encoding epidermal growth factor like domain]) is a novel secretory molecule that appears to act more specifically on osteoblasts than the BMPs, which can act on multiple cell types. From a molecular point of view, Nell-1 is directly regulated by runt-related transcription factor 2 (Runx2/Cbfa1), a master regulatory gene controlling bone formation. Previous studies showed that Nell-1 accelerates osteogenic differentiation in vitro and calvarial bone formation in vivo. We hypothesize that Nell-1 may also effectively form bone in spinal fusion. PURPOSE: Our primary aim was to assess if direct adenoviral gene delivery with Nell-1 in a demineralized bone matrix (DBM) carrier can improve spinal fusion in a rat model. Because adenoviral vectors allow for sustained growth factor delivery, they were used for initial feasibility testing before protein studies. STUDY DESIGN/SETTING: Two groups of 20 athymic rats underwent posterolateral intertransverse process spinal fusion at L4-L5 with implanted DBM carrier containing either adenovirus coding for Nell-1 (AdNell-1) or control, Lac Z (AdLacZ). No cells were implanted. The 20 rats were sacrificed at 6 weeks for evaluation of spinal fusion. METHODS: All animals underwent Faxitron radiographs at 2, 4, and 6 weeks, manual spine palpation at 6 weeks, and high-resolution micro computerized tomography (microCT) at 6 weeks. Spinal fusion rate was assessed by: 1) 6-week Faxitron images; 2) manual palpation by three independent observers; 3) microCT; and 4) histology. New bone formation was assessed by hematoxylin-eosin and Masson trichrome staining on decalcified, coronally sectioned spine segments. RESULTS: All differences achieved statistical significance. After 6 weeks, direct application of adenoviral Nell-1 in a DBM carrier achieved significantly higher rates of spinal fusion over Lac Z controls: 60% Nell-1 versus 20% Lac Z by manual palpation and 70% Nell-1 versus 20% Lac Z by microCT and histology. Histological assessment of bone quality and maturity revealed more mature, higher quality bone in all the Nell-1 treated specimens relative to Lac Z at 6 weeks. CONCLUSIONS: Spinal fusion is more accurately assessed by microCT and histology than manual palpation. Direct application of adenoviral Nell-1 in a DBM carrier achieved significantly higher rates of spinal fusion over Lac Z controls at 6 weeks. Direct application of adenoviral Nell-1 in a DBM carrier also achieved significantly higher rates of spinal fusion over other reports in the literature using direct adenoviral BMP application. Direct application of adenoviral BMP in an allograft carrier achieved 8% fusion for BMP-2 and 16% fusion for BMP-7 at 8 weeks. These results indicate that Nell-1 may be a potent osteoinductive molecule. In addition, the regulation of Nell-1 by the master bone regulatory gene, Runx2 suggests that Nell-1 may exert its effects more specifically in osteoblastic cells than BMPs which affect multiple cell types. Overall, Nell-1 may fulfil a current need for an osteoinductive factor.

Adenoviridae↗

PubMed2006

Nell-1 induces acrania-like cranioskeletal deformities during mouse embryonic development.

We previously reported NELL-1 as a novel molecule overexpressed during premature cranial suture closure in patients with craniosynostosis (CS). Nell-1 overexpression also results in premature suture closure/craniosynostosis in newborn transgenic mice. On a cellular level, increased levels of Nell-1 induce osteoblast differentiation and apoptosis. In this report, mice over-expressing Nell-1 were examined during embryonic development as well as shortly after birth for further analysis of craniofacial defects including neural tube defects (NTDs). The results demonstrated that overexpression of Nell-1 could induce acrania at relatively late gestation stage (E15.5) in mouse embryos, through massive apoptosis in calvarial osteoblasts and neural cells. The induced apoptosis was associated with an increase in Fas and Fas-L production. In addition, transgenic E15.5 and newborn transgenic mice with the CS phenotype displayed distortion of the chondrocranium associated with premature hypertrophy and increased apoptosis of chondrocytes. These findings were also verified in vitro with primary chondrocytes transduced with AdNell-1. In conclusion, Nell-1 overexpression can induce craniofacial anomalies associated with neural tube defects during embryonic development and may involve mechanisms of massive apoptosis associated with the Fas/Fas-L signaling pathway. NELL-1: used when describing the human gene; NELL-1: used when describing the human protein; Nell-1: used when describing the rodent gene; Nell-1: used when describing the rodent protein.

Animals↗

PubMed2006

Nell-1-induced bone regeneration in calvarial defects.

Many craniofacial birth defects contain skeletal components requiring bone grafting. We previously identified the novel secreted osteogenic molecule NELL-1, first noted to be overexpressed during premature bone formation in calvarial sutures of craniosynostosis patients. Nell-1 overexpression significantly increases differentiation and mineralization selectively in osteoblasts, while newborn Nell-1 transgenic mice significantly increase premature bone formation in calvarial sutures. In the current study, cultured calvarial explants isolated from Nell-1 transgenic newborn mice (with mild sagittal synostosis) demonstrated continuous bone growth and overlapping sagittal sutures. Further investigation into gene expression cascades revealed that fibroblast growth factor-2 and transforming growth factor-beta1 stimulated Nell-1 expression, whereas bone morphogenetic protein (BMP)-2 had no direct effect. Additionally, Nell-1-induced osteogenesis in MC3T3-E1 osteoblasts through reduction in the expression of early up-regulated osteogenic regulators (OSX and ALP) but induction of later markers (OPN and OCN). Grafting Nell-1 protein-coated PLGA scaffolds into rat calvarial defects revealed the osteogenic potential of Nell-1 to induce bone regeneration equivalent to BMP-2, whereas immunohistochemistry indicated that Nell-1 reduced osterix-producing cells and increased bone sialoprotein, osteocalcin, and BMP-7 expression. Insights into Nell-1-regulated osteogenesis coupled with its ability to stimulate bone regeneration revealed a potential therapeutic role and an alternative to the currently accepted techniques for bone regeneration.

Animals↗

PubMed2005

Nell-1 induced bone formation within the distracted intermaxillary suture.

Maxillary bone deficiencies, such as cleft palate and underdeveloped maxilla that require bone graft or regeneration after orthopedic or surgical expansion, pose a significant biomedical burden. Nell-1 is a secreted molecule that possesses chordin-like domains and induces cranial suture bone growth and osteoblast differentiation. To accelerate bone formation in acutely distracted palatal sutures, rat organ cultures were stimulated with Nell-1 or BMP-7 for 8 days in vitro. We hypothesized that Nell-1 stimulation to the distracted palatal suture would accelerate bone formation. Distracted palates of 4-week-old male rats were maintained in an organ culture system, and tissue was either unstimulated or stimulated with Nell-1 or BMP-7 for 8 days. MicroCT was conducted to quantitate bone formation, while alcian blue staining was conducted for cartilage localization. Immunohistochemistry of Sox9 for chondrocyte proliferation, type X collagen for hypertrophic cartilage in endochondral bone formation, and bone sialoprotein for bone formation was conducted to characterize the cellular mechanism of newly developed tissues. Distracted palates cultured in the presence of Nell-1 or BMP-7 produced statistically significantly (P < 0.05) more bone and cartilage within the intermaxillary suture, relative to unstimulated control samples. While both BMP-7 and Nell-1 induced similar bone formation in the distracted suture, BMP-7 induced both chondrocyte proliferation and differentiation, while Nell-1 accelerated chondrocyte hypertrophy and endochondral bone formation. While both Nell-1 and BMP-7 are effective in forming bone in the distracted palatal suture, they are suggested to have distinctively different mechanisms. The ability of Nell-1 to accelerate bone formation within the palate suture demonstrates the versatility of Nell-1 within the craniofacial complex as well as an exciting advance in palate suture defect healing.

Animals↗

PubMed2005

Evolving concepts in bone tissue engineering.

The field of tissue engineering integrates the latest advances in molecular biology, biochemistry, engineering, material science, and medical transplantation. Researchers in the developing field of regenerative medicine have identified bone tissue engineering as an attractive translational target. Clinical problems requiring bone regeneration are diverse, and no single regeneration approach will likely resolve all defects. Recent advances in the field of tissue engineering have included the use of sophisticated biocompatible scaffolds, new postnatal multipotent cell populations, and the appropriate cellular stimulation. In particular, synthetic polymer scaffolds allow for fast and reproducible construction, while still retaining biocompatible characteristics. These criteria relate to the immediate goal of determining the ideal implant. The search is becoming a reality with widespread availability of biocompatible scaffolds; however, the desired parameters have not been clearly defined. Currently, most research focuses on the use of bone morphogenetic proteins (BMPs), specifically BMP-2 and BMP-7. These proteins induce osteogenic differentiation in vitro, as well as bone defect healing in vivo. Protein-scaffold interactions that enhance BMP binding are of the utmost importance, since prolonged BMP release creates the most osteogenic microenvironment. Transition into clinical studies has had only mild success and relies on large doses of BMPs for bone formation. Advances within the field of bone tissue engineering will likely overcome these challenges and lead to more clinically relevant therapies.

Animals↗

PubMed2005

Gene expression profiling in the rat cranial suture.

Although many theories have attempted to explain the etiopathogenesis of premature cranial suture fusion, which results in craniosynostosis, recent studies have focused on the role of growth factors and receptors. Using a well-established model of cranial suture biology, the authors developed a novel approach to quantitatively analyze the gene expression profiles of candidate cranial suture growth factors and their receptors. We collected suture mesenchyme and adjacent osteogenic fronts from Sprague-Dawley rats at postnatal days 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 35. RNA was extracted from posterior frontal (PF) and sagittal (SAG) sutures, and reverse transcription-polymerase chain reaction (RT-PCR) was performed for cranial suture candidate cytokines BMP2, BMP3, BMP4, FGF-2, FGFR1, FGFR2, FGFR4, TGF-betaRI, TGF-betaRII, and TGF-betaRIII. The authors confirmed quantitative RT-PCR results with Southern and dot blot analyses. Suture growth factor and receptor expression levels changed significantly with time. Expression levels decreased toward baseline in the SAG suture by day 35. There was a marked difference in FGFR1, FGF-2, TGF-betaRI, and TGF-betaRII expression levels when comparing the fusing PF and nonfusing SAG sutures. Although FGF-2 ligand expression was low, FGF receptor 1 (FGFR1) levels were markedly elevated with a bimodal expression pattern in both PF and SAG similar to that of BMP2, BMP3, and BMP4. Although there were statistically significant differences in TGF-betaRI and TGF-betaRII expression in the PF and SAG sutures, TGF-betaRIII levels were unchanged. The authors report a novel approach to cranial suture growth factor/receptor profiling and confirm their results with standard analytic tools. The data confirm, quantify, and extend the results of previously published studies. By quantifying the gene expression profiles of normal cranial suture biology, we may begin to understand the aberrant growth factor cascades of craniosynostosis and devise targeted therapeutic interventions that can alter the course of this malady.

Animals↗

PubMed2005

Increased angiogenesis and expression of vascular endothelial growth factor during scarless repair.

Vascular endothelial growth factor (VEGF) is a dimeric heparin-binding glycoprotein that is a potent endothelial cell-specific mitogen with increased expression during adult cutaneous wound healing. VEGF activity is mediated by two receptors, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), which are expressed primarily in vascular endothelial cells. Initiation of profibrotic cytokine expression likely coordinates the transition from scarless healing to scar formation in fetal wounds. Angiogenesis is an important component of the scarring repair process, but the function of VEGF and degree of angiogenesis during scarless repair has not been investigated. We hypothesize that VEGF and its receptors are differentially expressed in scarless compared with scarring fetal wounds because VEGF is implicated in angiogenesis during skin development and adult wound healing. Excisional wounds were created on fetal rats at gestational ages 16.5 days (E16) and 18.5 days (E18) (term = 21.5 days). Wounds were harvested at 24 and 72 hours (n = 12 wounds per time point). Nonwounded fetal skin (E17, E19, and E21) was used as control. Reduced-cycle, specific-primer, reverse-transcriptase polymerase chain reaction was performed to determine the expression of VEGF and its receptors, VEGFR-1 and VEGFR-2. Wounds at 72 hours and fetal skin controls were examined under high-power microscopy for blood vessel counts. Unpaired two-tailed t test was used (p < 0.05 was considered significant). VEGF expression increased 2.4-fold (p < 0.001) during normal skin development from E17 to E19. In scarless wounds (E16), VEGF expression increased 2.8-fold (p < 0.02) at 72 hours. No increased expression occurred in the scarring wounds (E18). VEGFR-1 and VEGFR-2 expression increased over 2-fold during normal skin development from E17 to E21. However, each was down-regulated 30 to 50 percent in scarless (E16) and scarring (E18) wounds. There is a 2-fold increase in mean vessel counts per high-power field in scarless (E16) wounds at 72 hours compared with age-matched control skin (p < 0.02) and a 1.7-fold increase in mean vessel count in scarring fetal wounds (E18) compared with age-matched control skin (p < 0.05). There is no difference in the total number of vessels found in scarless versus scarring wounds or between 19.5-day versus 21.5-day fetal skin. VEGF and its receptors, VEGFR-1 and VEGFR-2, increase expression during skin development and dermal differentiation. VEGF expression quickly elevates during scarless compared with scarring repair, which likely contributes to the more rapid scarless fetal repair rate. Similar numbers of new ves-sels are formed during scarless and scarring fetal repair.

Animals↗

PubMed2003

Skin repair and scar formation: the central role of TGF-beta.

Wound healing is a complex process that we have only recently begun to understand. Central to wound repair is transforming growth factor beta (TGF-beta), a cytokine secreted by several different cell types involved in healing. TGF-beta has diverse effects, depending upon the tissue studied. This review focuses on healing in skin, particularly the phases of cutaneous wound repair and the role of TGF-beta in normal and impaired wound-healing models. It also explores TGF-beta activity in scarless foetal wound healing. Knowledge of TGF-beta function in scarless repair is critical to improving healing in clinical scenarios, such as diabetic wounds and hypertrophic scars.

Cicatrix↗

PubMed2003

Cephalometric evaluation of the craniofacial complex in patients treated with an intraoral distraction osteogenesis device: a preliminary report.

The purpose of this pilot study was to evaluate cephalometrically the efficacy of an intraoral distraction osteogenesis device in treating patients with unilateral mandibular hypoplasia. Six patients with hemifacial microsomia underwent unilateral mandibular distraction. Posteroanterior and 45 degrees lateral oblique cephalograms were measured, and changes in maxillary width and height, occlusal height, ramus height, mandibular length, and chin position were quantified. Measurements were taken preoperatively and postoperatively at 7 time points (T1-T7) over 2 years. Calculations for statistical significance were done to T6 for all patients and through T7 for 4 patients. The means and variances were calculated for the 6 cephalometric variables for each time point. The mean differences between treatment and control were calculated as well as analysis of variance. Mean differences between specific time periods were measured by pairwise comparison with significance determined at the 0.05 level of confidence. Statistical analysis was used for descriptive purposes only. The cephalometric data suggest that the intraoral distractor is as capable of lengthening hypoplastic mandibles as the initial extroral appliances. The bone lengthening appears stable, with the distracted side of the mandible maintaining a growth rate similar to the normal side. Immediately after distraction, transient improvements were noted in maxillary height, ramal height, and maxillary width. All patients demonstrated an immediate improvement in chin position toward the skeletal midline; however, after T4, menton appeared to be moving away from the midline over time.

Adolescent↗

PubMed2003

Ontogenetic transition in fetal wound transforming growth factor-beta regulation correlates with collagen organization.

Fetal rat skin transitions from scarless fetal-type repair to adult-type repair with scar between day 16 (E16) and day 18 (E18) of gestation (term = 21.5 days). Deficient transforming growth factor (TGF)-beta 1 and -beta 2 injury response has been proposed as a mechanism for scarless fetal-type repair. However, previous fetal studies have inconsistently reported the degree of TGF-beta induction after injury. To minimize developmental variables in fetal versus adult TGF-beta regulation, we narrowed our study to wounded fetal animals. We hypothesize that TGF-beta ligand and receptor expression will be differentially regulated during the transition from early gestation (E16) wounds manifesting scarless fetal-type repair to late gestation (E19) wounds manifesting adult-type repair with scar. In this study, decreased and rapidly cleared TGF-beta 1 and -beta 2 expression accompanied by increased and prolonged TGF-beta 3 levels in wounded E16 animals correlated with organized collagen deposition. In contrast, increased and prolonged TGF-beta 1 and -beta 2 expression accompanied by decreased and delayed TGF-beta 3 expression in wounded E19 animals correlated with disorganized collagen architecture. Similarly, expression of TGF-beta receptors type I and II were also increased or prolonged in E19 animals. Our results implicate increased TGF-beta 1, -beta 2, and decreased TGF-beta 3 expression, as well as increased type I and II receptor expression in late gestation fetal scar formation.

Animals↗

PubMed2003

Fetal wound healing current perspectives.

Early in gestation, fetal wounds are capable of healing scarlessly. Scarless healing in the fetus is characterized by regeneration of an organized dermis with normal appendages and by a relative lack of inflammation. Although there is a transition period between scarless and scar-forming repair, scarless healing also depends on wound size and the organ involved. The ability to heal scarlessly, furthermore, appears to be intrinsic to fetal skin. Unique characteristics of fetal fibroblasts, inflammatory cells, extra-cellular matrix, cytokine profile, and developmental gene regulation may be responsible for the scarless phenotype of early gestation fetal wounds. With the current knowledge, only minimal success has been achieved with the topical application of neutralizing antibodies, antisense oligonucleotides, and growth factors to improve wound-healing outcomes. Thus, further investigation into the mechanisms underlying scarless repair is crucial in order to devise more effective therapies for scar reduction and the treatment of cirrhosis, scleroderma, and other diseases of excessive fibrosis.

Animals↗

PubMed2003

Decreased expression of fibroblast and keratinocyte growth factor isoforms and receptors during scarless repair.

Fibroblast growth factors (FGFs) are a family of 21 cytokines with a broad spectrum of activities, including regulation of cell proliferation, differentiation, and migration. The various FGFs bind to one or more of four different tyrosine kinase receptor types. FGFs 1, 2, 5, 7, and 10 are up-regulated during adult cutaneous wound healing. However, the expression of FGFs during fetal skin development and scarless wound healing has not been characterized. It was hypothesized that differential expression of FGF isoforms and receptors occurs during fetal skin development and that this differential expression pattern may regulate the transition from scarless repair to healing with scar formation. Excisional wounds (2 mm) were created on fetal rats at gestational days 16.5 (scarless) (one wound per fetus, n = 36 fetuses) and 19.5 (scarring) (one wound per fetus, n = 36 fetuses). Wounds were harvested at 24, 48, and 72 hours. Survival until wound harvest ranged from 66 to 75 percent for the gestational day 16 fetuses, and from 83 to 92 percent for the gestational day 19 fetuses. Nonwounded fetal skin from littermates (n = 12 fetuses per wound harvest time point) was used as the control. Wounds/skins were pooled by harvest time point, and RNA was isolated from pooled wounds/skins. Reduced-cycle, specific-primer reverse transcriptase-polymerase chain reaction was performed to determine the expression of FGF isoforms 2, 5, 7, 9, and 10 and FGF receptors 1, 2, and 4 in wounds relative to unwounded skin.In unwounded fetal skin, FGF isoform 5 expression more than doubled at birth. FGF 10 expression doubled during the transition period. FGF 7 expression increased more than sevenfold at birth. Expression of FGF isoforms 2 and 9 did not change during late fetal skin development. The expression of FGF receptors 1, 2, and 4 increased at birth. After wounding, expression of FGF isoforms 7 and 10 was down-regulated in scarless wounds, whereas FGF receptor 2 expression decreased in both scarless and scar-forming wounds. Expression of FGF isoforms 5 and 9 did not change in scarless wounds. FGF receptor 2 expression was down-regulated in both scarless and scarring wounds, but at an earlier and more sustained level in scarless wounds. Receptor type 4 expression increased in scarring wounds, whereas type 1 expression did not change in either scarless or scarring wounds. These results demonstrate an overall down-regulation of FGF expression during scarless healing.

Animals↗

PubMed2003

Scarless fetal wounds are associated with an increased matrix metalloproteinase-to-tissue-derived inhibitor of metalloproteinase ratio.

In contrast to adult cutaneous wounds, early fetal wounds heal scarlessly. Fetal rat skin transitions from scarless repair to healing, with scar formation between days 16.5 (E16) and 18.5 (E18) of gestation. Term gestation is 21.5 days. The composition of the extracellular matrix in fetal skin and wounds differs from that of the adult. Matrix metalloproteinases (MMPs) and their tissue-derived inhibitors (TIMPs) determine the architecture of the extracellular matrix. The authors hypothesized that differential expression of MMPs and TIMPs occurs during the ontogenetic transition to scar-forming repair in fetal skin and wounds. Full-thickness, excisional wounds (2 mm) were created on the dorsum of E16 (n = 42 fetuses) and E19 fetal rats (n = 42 fetuses). Wounds were harvested at 24, 48, and 72 hours. Nonwounded skin from littermates was also harvested as controls. Six E16 and E19 wounds were fixed 72 hours after injury, stained with hematoxylin and eosin, and examined by light microscopy. RNA was isolated from the remaining wounds and skin, and a reduced-cycle, primer-specific, reverse-transcriptase polymerase chain reaction was performed to semiquantitatively determine relative gene expression of MMP-1, MMP-2, MMP-7, MMP-9, and MMP-14 and of TIMP-1, TIMP-2, and TIMP-3. Significance was determined by unpaired two-tailed t test (p < 0.05) and analysis of variance. In both E16 and E19 wounds, reepithelialization was complete by 72 hours. E16 wounds healed scarlessly, whereas E19 wounds healed with scar. During late gestation, skin expression of MMP-1 and MMP-14 (membrane type-1 MMP) doubled, whereas MMP-2 expression increased nearly 50-fold. Levels of MMP-7 and MMP-9 were unchanged in developing skin. As for the TIMPs, skin expression of TIMP-2 increased more than four-fold, whereas TIMP-1 and TIMP-3 expression was unchanged. In both scarless and scarring wounds, up-regulation of MMP-1 and MMP-9 occurred. However, the maximal increase in MMP-1 and MMP-9 expression occurred much more rapidly and was much greater in the scarless E16 wounds (28-fold versus 23-fold for MMP-1 and 18-fold versus nine-fold for MMP-9). Unchanged in scarless wounds, MMP-2 levels decreased more than three-fold in scarring wounds. MMP-14 (membrane type-1 MMP) expression increased three-fold in scarless wounds but was unchanged in scarring wounds. In contrast, TIMP-1 and TIMP-3 expression in E19 scarring wounds increased six-fold and four-fold, respectively. MMP-7 and TIMP-2 expression did not change in response to injury. E16 scarless wounds have greater MMP relative to TIMP expression than E19 scarring wounds. This favors extracellular matrix turnover, facilitates migration of fetal cells, and promotes scarless repair.

Animals↗

PubMed2003

Overexpression of Nell-1, a craniosynostosis-associated gene, induces apoptosis in osteoblasts during craniofacial development.

UNLABELLED: We studied the cellular function of Nell-1, a craniosynostosis-related gene, in craniofacial development. Nell-1 modulates calvarial osteoblast differentiation and apoptosis pathways. Nell-1 overexpression disrupts these pathways resulting in craniofacial anomalies such as premature suture closure. INTRODUCTION: Craniosynostosis (CS), one of the most common congenital craniofacial deformities, is the premature closure of cranial sutures. Previously, we reported NELL-1 as a novel molecule overexpressed during premature cranial suture closure in patients with CS. Nell-1 overexpression induced calvarial overgrowth and resulted in premature suture closure in a rodent model. On a cellular level, Nell-1 is suggested to promote osteoblast differentiation. MATERIALS AND METHODS: Different levels of Nell-1 were introduced into osteoblastic cells by viral infection and recombinant protein. Apoptosis and gene expression assays were performed. Mice overexpressing Nell-1 were examined for apoptosis. RESULTS: In this report, we further showed that overexpression of Nell-1 induced apoptosis along with modulation of apoptosis-related genes. The induction of apoptosis by Nell-1 was observed only in osteoblastic cells and not in NIH3T3 or primary fibroblasts. The CS mouse model overexpressing Nell-1 showed increased levels of apoptosis in the calvaria. CONCLUSION: We show that Nell-1 expression modulates calvarial osteoblast differentiation and apoptosis pathways. Nell-1 overexpression disrupts these pathways resulting in craniofacial anomalies such as premature suture closure.

Animals↗

PubMed2002

The identification of novel wound-healing genes through differential display.

Effective methods to identify novel genes in complicated dynamic tissue processes are needed in molecular biology research. Traditional techniques primarily target known genes and are inefficient in the pursuit of unknown genes. Here we describe the use of a modified differential display polymerase chain reaction (DD-PCR) protocol for the identification of genes differentially expressed in wound healing. Full-thickness dorsal wounds were made on 35 adult rats, followed by wound harvest at 12 hours, 24 hours, 3 days, 5 days, 7 days, 10 days, and 14 days after injury. Modified DD-PCR was performed and gene fragments displaying definite changes during wound healing were cloned and sequenced. Gene fragments from DD-PCR were compared with available gene bank database sequences. Specific primer PCR was used to confirm DD-PCR expression patterns. As a result, over 1000 gene fragments were amplified by DD-PCR, 35 of which demonstrated distinct differences during repair. Cloning and sequencing of 13 of these gene fragments revealed that some were homologous to several characterized genes with previously unsuspected roles in repair, whereas others were completely novel genes with no known function. Specific primer PCR further confirmed expression of six of these 13 gene fragments. Only one of the 13 cloned fragments, later identified as interleukin-1beta, had well-recognized associations with tissue injury. Other fragments corresponded to various genes involved in cellular processes such as differentiation, proliferation, exocytosis, and myofibril assembly. No prior studies have linked them to wound healing. We have demonstrated that modified DD-PCR can be used to effectively identify novel genes differentially expressed during repair. Because DD-PCR allows for the simultaneous amplification of multiple arbitrary transcripts, it is a powerful genetic screening tool for complicated dynamic tissue processes, particularly when multiple, limited-sized samples are involved.

Animals↗

PubMed2002

Confocal microscopic analysis of scarless repair in the fetal rat: defining the transition.

Fetal wounds pass from scarless repair to healing with scar formation during gestation. This transition depends on both the size of the wound and the gestational age of the fetus. This study defines the transition period in the fetal rat model and provides new insight into scarless collagen wound architecture by using confocal microscopy. A total of 16 pregnant Sprague-Dawley rats were operated on. Open full-thickness wounds, 2 mm in diameter, were created on fetal rats at gestational ages 14.5 days (E14; n = 10), 16.5 days (E16; n = 42), and 18.5 days (E18; n = 42) (term = 21.5 days). Wounds were harvested at 24 (n = 18 per gestational age) and 72 hours (n = 24 per gestational age). Skin at identical gestational ages to wound harvest was used for controls. The wounds were fixed and stained with hematoxylin and eosin, antibody to type I collagen, and Sirius red for confocal microscopic evaluation. No E14 rat fetuses survived to wound harvest. Wounds created on E16 fetal rats healed completely and without scarring. E16 fetal rat hair follicle formation and collagen architecture was similar to that of normal, nonwounded skin. Wounds created on E18 fetal rats demonstrated slower healing; only 50 percent were completely healed at 72 hours compared with 100 percent of the E16 fetal rat wounds at 72 hours. Furthermore, the E18 wounds healed with collagen scar formation and without hair follicle formation. Confocal microscopy demonstrated that the collagen fibers were thin and arranged in a wispy pattern in E16 fetal rat wounds and in nonwounded dermis. E18 fetal rat wounds had thickened collagen fibers with large interfiber distances. Two-millimeter excisional E16 fetal rat wounds heal without scar formation and with regeneration of normal dermal and epidermal appendage architecture. E18 fetal rat wounds heal in a pattern similar to that of adult cutaneous wounds, with scar formation and absence of epidermal appendages. Confocal microscopy more clearly defined the dermal architecture in normal skin, scarless wounds, and scars. These data further define the transition period in the fetal rat wound model, which promises to be an effective system for the study of in vivo scarless wound healing.

Animals↗

PubMed2002

Craniosynostosis in transgenic mice overexpressing Nell-1.

Previously, we reported NELL-1 as a novel molecule overexpressed during premature cranial suture closure in patients with craniosynostosis (CS), one of the most common congenital craniofacial deformities. Here we describe the creation and analysis of transgenic mice overexpressing Nell-1. Nell-1 transgenic animals exhibited CS-like phenotypes that ranged from simple to compound synostoses. Histologically, the osteogenic fronts of abnormally closing/closed sutures in these animals revealed calvarial overgrowth and overlap along with increased osteoblast differentiation and reduced cell proliferation. Furthermore, anomalies were restricted to calvarial bone, despite generalized, non-tissue-specific overexpression of Nell-1. In vitro, Nell-1 overexpression accelerated calvarial osteoblast differentiation and mineralization under normal culture conditions. Moreover, Nell-1 overexpression in osteoblasts was sufficient to promote alkaline phosphatase expression and micronodule formation. Conversely, downregulation of Nell-1 inhibited osteoblast differentiation in vitro. In summary, Nell-1 overexpression induced calvarial overgrowth resulting in premature suture closure in a rodent model. Nell-1, therefore, has a novel role in CS development, perhaps as part of a complex chain of events resulting in premature suture closure. On a cellular level, Nell-1 expression may modulate and be both sufficient and required for osteoblast differentiation.

Adenoviridae↗