"Effect of micro-osteoperforations. Authors' response" [Letter]Alikhani, Mani; Raptis, Markos; Zoldan, Billie; Sangsuwon, Chinapa; Lee, Yoo B; Alyami, Bandar; Corpodian, Corey; Barrera, Luz M; Alansari, Sarah; Khoo, Edmund; TEIXEIRA, CRISTINA
"Effect of micro-osteoperforations on the rate of tooth movement"Alikhani, Mani; Raptis, Markos; Zoldan, Billie; Sangsuwon, Chinapa; Lee, Yoo B; Alyami, Bandar; Corpodian, Corey; Barrera, Luz M; Alansari, Sarah; Khoo, Edmund; TEIXEIRA, CRISTINA
INTRODUCTION: Our objectives were to study the effect of micro-osteoperforations on the rate of tooth movement and the expression of inflammatory markers. METHODS: Twenty adults with Class II Division 1 malocclusion were divided into control and experimental groups. The control group did not receive micro-osteoperforations, and the experimental group received micro-osteoperforations on 1 side of the maxilla. Both maxillary canines were retracted, and movement was measured after 28 days. The activity of inflammatory markers was measured in gingival crevicular fluid using an antibody-based protein assay. Pain and discomfort were monitored with a numeric rating scale. RESULTS: Micro-osteoperforations significantly increased the rate of tooth movement by 2.3-fold; this was accompanied by a significant increase in the levels of inflammatory markers. The patients did not report significant pain or discomfort during or after the procedure, or any other complications. CONCLUSIONS: Micro-osteoperforation is an effective, comfortable, and safe procedure to accelerate tooth movement and significantly reduce the duration of orthodontic treatment..
"Evaluation of osteogenic cell culture and osteogenic/peripheral blood mononuclear human cell co-culture on modified titanium surfaces"Moura, Camilla G; Souza, Maria A; Kohal, Ralf J; Dechichi, Paula; Zanetta-Barbosa, Darceny; Jimbo, Ryo; TEIXEIRA, CRISTINA C; Teixeira, Hellen S; Tovar, Nick; Coelho, Paulo G
This study aimed to determine the effect of a bioactive ceramic coating on titanium in the nanothickness range on human osteogenic cells, peripheral blood mononuclear cells (PBMC) and on osteogenic cells co-cultured with PBMC without exogenous stimuli. Cell viability, proliferation, adhesion, cytokine release (IL1beta, TGFbeta1, IL10 and IL17) and intracellular stain for osteopontin and alkaline phosphatase were assessed. Morphologic evaluation showed smaller and less spread cell aspects in co-culture relative to osteogenic cell culture. Cell viability, proliferation and adhesion kinetics were differently influenced by surface texture/chemistry in culture versus co-culture. Cytokine release was also influenced by the interaction between mononuclear and osteogenic cells (mediators released by mononuclear cells acted on osteogenic cells and vice versa). In general, 'multi-cell type' interactions played a more remarkable role than the surface roughness or chemistry utilized on the in vitro cellular events related to initial stages of bone formation..
"Chondrogenic ATDC5 cells: An optimised model for rapid and physiological matrix mineralisation"Newton, P T; Staines, K A; Spevak, L; Boskey, A L; TEIXEIRA, C C; Macrae, V E; Canfield, A E; Farquharson, C
GRANTS:AR046121/AR/NIAMS NIH HHS/United States;Biotechnology and Biological Sciences Research Council/United Kingdom
The development of chondrogenic cell lines has led to major advances in the understanding of how chondrocyte differentiation is regulated, and has uncovered many signalling pathways and gene regulatory mechanisms required to maintain normal function. ATDC5 cells are a well established in vitro model of endochondral ossification; however, current methods are limited for mineralisation studies. In this study we demonstrate that culturing cells in the presence of ascorbic acid and 10 mM beta-glycerophosphate (betaGP) significantly increases the rate of extracellular matrix (ECM) synthesis and reduces the time required for mineral deposition to occur to 15 days of culture. Furthermore, the specific expression patterns of Col2a1 and Col10a1 are indicative of ATDC5 chondrogenic differentiation. Fourier transform-infrared spectroscopy analysis and transmission electron microscopy (TEM) showed that the mineral formed by ATDC5 cultures is similar to physiological hydroxyapatite. Additionally, we demonstrated that in cultures with betaGP, the presence of alkaline phosphatase (ALP) is required for this mineralisation to occur, further indicating that chondrogenic differentiation is required for ECM mineralisation. Together, these results demonstrate that when cultured in the presence of ascorbic acid and 10 mM betaGP, ATDC5 cells undergo chondrogenic differentiation and produce a physiological mineralised ECM from Day 15 of culture onwards. The rapid and novel method for ATDC5 culture described in this study is a major improvement compared with currently published methods and this will prove vital in the pursuit of underpinning the molecular mechanisms responsible for poor linear bone growth observed in a number of chronic diseases such as cystic fibrosis, chronic kidney disease, rheumatological conditions and inflammatory bowel disease..
"Changes in matrix protein gene expression associated with mineralization in the differentiating chick limb-bud micromass culture system"TEIXEIRA, CRISTINA C; Xiang, Jenny; Roy, Rani; Kudrashov, Valery; Binderman, Itzhak; Mayer-Kuckuk, Philipp; Boskey, Adele L
GRANTS:AR037661/AR/NIAMS NIH HHS/United States;R01 AR037661/AR/NIAMS NIH HHS/United States
Chick limb-bud mesenchymal stem cells plated in high density culture in the presence of 4 mM inorganic phosphate and vitamin C differentiate and form a mineralizable matrix, resembling that of the chick growth plate. To further elucidate the mechanism that allows these cultures to form physiologic hydroxyapatite deposits, and how the process can be manipulated to gain insight into mineralization mechanisms, we compared gene expression in mineralizing (with 4 mM inorganic phosphate) and non-mineralizing cultures (containing only 1 mM inorganic phosphate) at the start of mineralization (day 11) and after mineralization reached a plateau (day 17) using a chick specific microarray. Based on replicate microarray experiments and K-cluster analysis, several genes associated with the mineralization process were identified, and their expression patterns confirmed throughout the culture period by quantitative RT-PCR. The functions of bone morphogenetic protein 1, BMP1, dentin matrix protein 1, DMP1, the sodium phosphate co-transporter, NaPi IIb, matrix metalloprotease 13. MMP-13, and alkaline phosphatase, along with matrix protein genes (type X collagen, bone sialoprotein, and osteopontin) usually associated with initiation of mineralization are discussed. J. Cell. Biochem. 112: 607-613, 2011. (c) 2010 Wiley-Liss, Inc.
"The effect of a nanothickness coating on rough titanium substrate in the osteogenic properties of human bone cells"Moura, Camilla C G; Souza, Maria A; Dechichi, Paula; Zanetta-Barbosa, Darceny; TEIXEIRA, CRISTINA C; Coelho, Paulo G
This study evaluated the effect of a bioactive ceramic coating, in the nanothickness range, onto a moderately rough surface on the osteogenic behavior of human bone cells. The cells were harvested from the mandibular mental region and were cultured over Ti-6Al-4V disks of different surfaces: as-machined (M), alumina-blasted/acid etched (AB/AE), and alumina-blasted/acid-etched + 300-500 nm thickness amorphous Ca- and P-based coating obtained by ion beam-assisted deposition (Nano). The culture was then evaluated regarding cell viability, adhesion, morphology, immunolocalization of osteopontin (OPN) and alkaline phosphatase (ALP). The results showed that the surface treatment did not interfere with cell viability. At 1 day, AB/AE and Nano showed higher adhesion than the M surface (p < 0.001). Higher adhesion was observed for the M than the Nano surface at 7 days (p < 0.005). The percentage of cells showing intracellular labeling for OPN at day 1 was significantly higher for the Nano compared to M surface (p < 0.03). The percentage of ALP intracellular labeling at 7 days was significantly higher for the AB/AE compared to the M surface (p < 0.0065); no differences were detected at 14 days. Our results suggest that the presence of a thin bioactive ceramic coating on a rough substrate did not favor the events related to in vitro osteogenesis. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010..
"An improved collagen scaffold for skeletal regeneration"Oliveira, Serafim M; Ringshia, Rushali A; Legeros, Racquel Z; Clark, Elizabeth; Yost, Michael J; Terracio, Louis; TEIXEIRA, CRISTINA C
GRANTS:K08 DE017426-04S1/DE/NIDCR NIH HHS/United States
Bone repair and regeneration is one of the most extensively studied areas in the field of tissue engineering. All of the current tissue engineering approaches to create bone focus on intramembranous ossification, ignoring the other mechanism of bone formation, endochondral ossification. We propose to create a transient cartilage template in vitro, which could serve as an intermediate for bone formation by the endochondral mechanism once implanted in vivo. The goals of the study are (1) to prepare and characterize type I collagen sponges as a scaffold for the cartilage template, and (2) to establish a method of culturing chondrocytes in type I collagen sponges and induce cell maturation. Collagen sponges were generated from a 1% solution of type I collagen using a freeze/dry technique followed by UV light crosslinking. Chondrocytes isolated from two locations in chick embryo sterna were cultured in these sponges and treated with retinoic acid to induce chondrocyte maturation and extracellular matrix deposition. Material strength testing as well as microscopic and biochemical analyzes were conducted to evaluate the properties of sponges and cell behavior during the culture period. We found that our collagen sponges presented improved stiffness and supported chondrocyte attachment and proliferation. Cells underwent maturation, depositing an abundant extracellular matrix throughout the scaffold, expressing high levels of type X collagen, type I collagen and alkaline phosphatase. These results demonstrate that we have created a transient cartilage template with potential to direct endochondral bone formation after implantation..
"F-spondin regulates chondrocyte terminal differentiation and endochondral bone formation"Palmer, Glyn D; Piton, Alejandro H; Thant, Lwin Mon; Oliveira, Serafim M; D'Angelo, Marina; Attur, Mukundan G; Abramson, Steven B; TEIXEIRA, CRISTINA C
GRANTS:K08DE017426/DE/NIDCR NIH HHS/United States;R01 AR054817/AR/NIAMS NIH HHS/United States;R01 AR054817-01A2/AR/NIAMS NIH HHS/United States;R01-AR054817/AR/NIAMS NIH HHS/United States
This study examines the role of F-spondin, an extracellular matrix protein of osteoarthritic cartilage, during chondrocyte maturation in embryonic growth plate cartilage. In chick tibia, F-spondin expression localized to the hypertrophic and calcified zones of the growth plate. Functional studies using tibial organ cultures indicated that F-spondin inhibited ( approximately 35%, p = 0.02), and antibodies to F-spondin increased ( approximately 30%, p < 0.1) longitudinal limb growth relative to untreated controls. In cell cultures, induction of chondrocyte maturation, by retinoic acid (RA) or transforming growth factor (TGF)-beta treatment led to a significant upregulation of F-spondin (p < 0.05). F-spondin transfection increased mineral deposition, alkaline phosphatase (AP) and matrix metalloproteinase (MMP)-13 mRNA levels (p < 0.05), and AP activity following RA stimulation, compared to mock transfected controls. Using AP as a differentiation marker we then investigated the mechanism of F-spondin promaturation effects. Blocking endogenous F-spondin via its thrombospondin (TSR) domain inhibited RA induced AP activity 40% compared to controls (p < 0.05). The stimulatory effect of F-spondin on AP expression was also inhibited following depletion of TGF-beta from culture supernatants. Our findings indicate that F-spondin is expressed in embryonic cartilage, where it has the capacity to enhance chondrocyte terminal differentiation and mineralization via interactions in its TSR domain and TGF-beta dependent pathways..
"Personalized implant therapy: new perspectives in bone remodeling assessment"Razzouk, Sleiman; TEIXEIRA, CRISTINA
Current approaches for immediately loading dental implants rely upon clinical trials and evidence-based practice. Despite their wide utility, these studies have limited predictability because they ignore a whole set of parameters, mainly the environmental parameters that have increasingly influenced the bone biological response. Bone is a very dynamic tissue and its reaction to surgical and functional implant procedures is variable; therefore, bone remodeling must be a key factor in any clinical decision, and a need for reliable tests to evaluate this process is an eventual challenge to implant success. New advances in medicine based upon individual genomic, proteomic and metabolomic studies incorporate the impact of environmental elements, permitting a better targeting of implant therapy. Previously, we proposed a new clinical concept of implant therapy based on personalized bone turnover. Here, we elaborate on current tests and future 'omics' biotechnologies to assess the turnover process, hence, providing a realistic approach to individual evaluations of bone remodeling.
"Changes in matrix protein gene expression associated with mineralization in the differentiating chick limb-bud micromass culture system"TEIXEIRA CC; Xiang J; Roy R; Kudrashev V; Binderman I; Mayer-Kuckuk P; Boskey AL
Chick limb-bud mesenchymal stem cells plated in high density culture in the presence of 4 mM inorganic phosphate and vitamin C differentiate and form a mineralizable matrix, resembling that of the chick growth plate . To further elucidate the mechanism that allows these cultures to form physiologic hydroxyapatite deposits, and how the process can be manipulated to gain insight into mineralization mechanisms, we compared gene expression in mineralizing (with 4 mM inorganic phosphate) and non-mineralizing cultures (containing only 1 mM inorganic phosphate) at the start of mineralization (day 11) and after mineralization reached a plateau (day 17) using a chick specific micro-array. Based on replicate micro-array experiments and K-cluster analysis, several genes associated with the mineralization process were identified, and their expression patterns confirmed throughout the culture period by quantitative RT-PCR. The functions of bone morphogenetic protein 1, BMP1, dentin matrix protein 1, DMP1, the sodium phosphate co-transporter, NaPi IIb, matrix metalloprotease 13. MMP-13, and alkaline phosphatase, along with matrix protein genes (type X collagen, bone sialoprotein, and osteopontin) usually associated with initiation of mineralization are discussed. (c) 2010 Wiley-Liss, Inc.
"Cytokine expression and accelerated tooth movement"TEIXEIRA, C C; Khoo, E; Tran, J; Chartres, I; Liu, Y; Thant, L M; Khabensky, I; Gart, L P; Cisneros, G; Alikhani, M
GRANTS:AR046121/AR/NIAMS NIH HHS/United States;K08DE017426/DE/NIDCR NIH HHS/United States;R03DE019499/DE/NIDCR NIH HHS/United States
It has been shown that inhibiting the expression of certain cytokines decreases the rate of tooth movement. Here, we hypothesized that stimulating the expression of inflammatory cytokines, through small perforations of cortical bone, increases the rate of bone remodeling and tooth movement. Forty-eight rats were divided into 4 groups: 50-cN force applied to the maxillary first molar (O), force application plus soft tissue flap (OF), force application plus flap plus 3 small perforations of the cortical plate (OFP), and a control group (C). From the 92 cytokines studied, the expression of 37 cytokines increased significantly in all experimental groups, with 21 cytokines showing the highest levels in the OFP group. After 28 days, micro-computed tomography, light and fluorescent microscopy, and immunohistochemistry demonstrated higher numbers of osteoclasts and bone remodeling activity in the OFP group, accompanied by generalized osteoporosity and increased rate of tooth movement.
"Foxo1, a novel regulator of osteoblast differentiation and skeletogenesis"TEIXEIRA, CRISTINA C; Liu, Yuexun; Thant, Lwin M; Pang, Jason; Palmer, Glyn; Alikhani, Mani
GRANTS:5K08DE017426/DE/NIDCR NIH HHS/United States;AR046121/AR/NIAMS NIH HHS/United States;R03 DE019499-01A1/DE/NIDCR NIH HHS/United States;R03 DE019499-02/DE/NIDCR NIH HHS/United States;R03DE019499/DE/NIDCR NIH HHS/United States
Skeletogenesis depends on the activity of bone-forming cells derived from mesenchymal cells. The pathways that control mesenchymal cell differentiation are not well understood. We propose that Foxo1 is an early molecular regulator during mesenchymal cell differentiation into osteoblasts. In mouse embryos, Foxo1 expression is higher in skeletal tissues, while Foxo1 silencing has a drastic impact on skeletogenesis and craniofacial development, specially affecting pre-maxilla, nasal bone, mandible, tibia, and clavicle. Similarly, Foxo1 activity and expression increase in mouse mesenchymal cells under the influence of osteogenic stimulants. In addition, silencing Foxo1 blocks the expression of osteogenic markers such as Runx2, alkaline phosphatase, and osteocalcin and results in decreased culture calcification even in the presence of strong osteogenic stimulants. Conversely, the expression of these markers increases significantly in response to Foxo1 overexpression. One mechanism through which Foxo1 affects mesenchymal cell differentiation into osteoblasts is through regulation of a key osteogenic transcription factor, Runx2. Indeed, our results show that Foxo1 directly interacts with the promoter of Runx2 and regulates its expression. Using a tibia organ culture model, we confirmed that silencing Foxo1 decreases the expression of Runx2 and impairs bone formation. Furthermore, our data reveals that Runx2 and Foxo1 interact with each other and cooperate in the transcriptional regulation of osteoblast markers. In conclusion, our in vitro, ex vivo, and in vivo results strongly support the notion that Foxo1 is an early molecular regulator in the differentiation of mesenchymal cells into osteoblast..
"F-spondin, a neuroregulatory protein, is up-regulated in osteoarthritis and regulates cartilage metabolism via TGF-beta activation"Attur, Mukundan G; Palmer, Glyn D; Al-Mussawir, Hayf E; Dave, Mandar; TEIXEIRA, CRISTINA C; Rifkin, Daniel B; Appleton, C Thomas G; Beier, Frank; Abramson, Steven B
GRANTS:CA034282/CA/NCI NIH HHS/United States;R01-AR054817/AR/NIAMS NIH HHS/United States;T32-AR007176/AR/NIAMS NIH HHS/United States
In osteoarthritis (OA) articular chondrocytes undergo phenotypic changes culminating in the progressive loss of cartilage from the joint surface. The molecular mechanisms underlying these changes are poorly understood. Here we report enhanced (approximately 7-fold) expression of F-spondin, a neuronal extracellular matrix glycoprotein, in human OA cartilage (P<0.005). OA-specific up-regulation of F-spondin was also demonstrated in rat knee cartilage following surgical menisectomy. F-spondin treatment of OA cartilage explants caused a 2-fold increase in levels of the active form of TGF-beta1 (P<0.01) and a 10-fold induction of PGE2 (P<0.005) in culture supernatants. PGE2 induction was found to be dependent on TGF-beta and the thrombospondin domain of the F-spondin molecule. F-spondin addition to cartilage explant cultures also caused a 4-fold increase in collagen degradation (P<0.05) and a modest reduction in proteoglycan synthesis (approximately 20%; P<0.05), which were both TGF-beta and PGE2 dependent. F-spondin treatment also led to increased secretion and activation of MMP-13 (P<0.05). Together these studies identify F-spondin as a novel protein in OA cartilage, where it may act in situ at lesional areas to activate latent TGF-beta and induce cartilage degradation via pathways that involve production of PGE2..
"Engineering endochondral bone: in vitro studies"Oliveira, Serafim M; Amaral, Isabel F; Barbosa, Mario A; TEIXEIRA, CRISTINA C
GRANTS:5K08DE017426/DE/NIDCR NIH HHS/United States;AR046121/AR/NIAMS NIH HHS/United States;K08 DE017426-01A1/DE/NIDCR NIH HHS/United States
Chitosan scaffolds have been shown to possess biological and mechanical properties suitable for tissue engineering and clinical applications. In the present work, chitosan sponges were evaluated regarding their ability to support cartilage cell proliferation and maturation, which are the first steps in endochondral bone formation. Chitosan sponges were seeded with chondrocytes isolated from chicken embryo sterna. Chondrocyte/chitosan constructs were cultured for 20 days, and treated with retinoic acid (RA) to induce chondrocyte maturation and matrix synthesis. At different time points, samples were collected for microscopic, histological, biochemical, and mechanical analyses. Results show chondrocyte attachment, proliferation, and abundant matrix synthesis, completely obliterating the pores of the sponges. RA treatment caused chondrocyte hypertrophy, characterized by the presence of type X collagen in the extracellular matrix and increased alkaline phosphatase activity. In addition, hypertrophy markedly changed the mechanical properties of the chondrocyte/chitosan constructs. In conclusion, we have developed chitosan sponges with adequate pore structure and mechanical properties to serve as a support for hypertrophic chondrocytes. In parallel studies, we have evaluated the ability of this mature cartilage scaffold to induce endochondral ossification..
"Engineering endochondral bone: in vivo studies"Oliveira, Serafim M; Mijares, Dindo Q; Turner, Gloria; Amaral, Isabel F; Barbosa, Mario A; TEIXEIRA, CRISTINA C
GRANTS:5K08DE017426/DE/NIDCR NIH HHS/United States;K08 DE017426-01A1/DE/NIDCR NIH HHS/United States;K08 DE017426-02/DE/NIDCR NIH HHS/United States;K08 DE017426-03/DE/NIDCR NIH HHS/United States
The use of biomaterials to replace lost bone has been a common practice for decades. More recently, the demands for bone repair and regeneration have pushed research into the use of cultured cells and growth factors in association with these materials. Here we report a novel approach to engineer new bone using a transient cartilage scaffold to induce endochondral ossification. Chondrocyte/chitosan scaffolds (both a transient cartilage scaffold-experimental-and a permanent cartilage scaffold-control) were prepared and implanted subcutaneously in nude mice. Bone formation was evaluated over a period of 5 months. Mineralization was assessed by Faxitron, micro computed tomography, backscatter electrons, and Fourier transform infrared spectroscopy analyses. Histological analysis provided further information on tissue changes in and around the implanted scaffolds. The deposition of ectopic bone was detected in the surface of the experimental implants as early as 1 month after implantation. After 3 months, bone trabeculae and bone marrow cavities were formed inside the scaffolds. The bone deposited was similar to the bone of the mice vertebra. Interestingly, no bone formation was observed in control implants. In conclusion, an engineered transient cartilage template carries all the signals necessary to induce endochondral bone formation in vivo..
"F-spondin, a neuroregulatory protein, is upregulated in human and surgically-induced osteoarthritis: Evidence for regulation of cartilage metabolism via latent tgf-b1 activation" [Meeting Abstract]Attur, M; Palmer, G; Al-Mussawir, HE; Rifkin, DB; TEIXEIRA, CC; Appleton, CTG; Beier, F; Abramson, SB
"Nitric oxide, C-type natriuretic peptide and cGMP as regulators of endochondral ossification"TEIXEIRA, CRISTINA C; Agoston, Hanga; Beier, Frank
GRANTS:5K08DE017426/DE/NIDCR NIH HHS/United States;K08 DE017426-01A1/DE/NIDCR NIH HHS/United States;K08 DE017426-02/DE/NIDCR NIH HHS/United States
Coordinated proliferation and differentiation of growth plate chondrocytes is required for endochondral bone growth, but the mechanisms and pathways that control these processes are not completely understood. Recent data demonstrate important roles for nitric oxide (NO) and C-type natriuretic peptide (CNP) in the regulation of cartilage development. Both NO and CNP stimulate the synthesis of cGMP and thus the activation of common downstream pathways. One of these downstream mediators, cGMP-dependent kinase II (cGKII), has itself been shown to be essential for normal endochondral bone formation. This review summarizes our knowledge of the roles and mechanisms of NO, CNP and cGKII signaling in cartilage and endochondral bone development..
"Apoptosis of Growth Plate Chondrocytes Occurs through a Mitochondrial Pathway"TEIXEIRA, CRISTINA C; Costas, Aida P Padron; Nemelivsky, Yelena
Objective: To determine the role of mitochondria in chondrocyte apoptosis induced by inorganic phosphate (Pi). Materials and Methods: Chondrocytes isolated from the growth plates of chick embryo tibia were treated with Pi in serum-free media; chondrocyte viability, mitochondrial membrane potential, cytochrome c release from mitochondria, caspase 3 activity, endonuclease activity, and DNA fragmentation were investigated. Results: Exposure to Pi for 24 hours induced apoptosis in growth plate chondrocytes through a pathway that involved loss of mitochondrial function, release of cytochrome c into the cytoplasm, increases in caspase 3 and endonuclease activities, and fragmentation of DNA. Conclusions: This study suggests that mitochondria are important players in Pi-induced apoptosis.
"Biphasic calcium phosphate: a scaffold for growth plate chondrocyte maturation"TEIXEIRA, CRISTINA C; Nemelivsky, Yelena; Karkia, Claudia; Legeros, Racquel Z
While skeletal development can occur by either intramembranous or endochondral bone formation, all current tissue engineering approaches for bone repair and regeneration try to mimic intramembranous ossification. In this study, we propose to create an in vitro cartilage template as the transient model for in vivo endochondral bone formation. The goals of this study are to (1) establish a method of growing chondrocytes in a well-characterized macroporous biphasic calcium phosphate (MBCP) scaffold and (2) induce maturation of chondrocytes grown in the MBCP scaffold. Chondrocytes isolated from chick embryonic tibia were grown on MBCP particles and treated with retinoic acid to induce chondrocyte maturation and extracellular matrix deposition. Chondrocytes were observed to attach and proliferate on the MBCP scaffold. The thickness of the chondrocyte and extracellular matrix layer increased in the presence of the retinoid. Alkaline phosphatase activity and expression, proteoglycans synthesis, cbfa1 and type I collagen mRNA levels also increased in the presence of retinoic acid. These results demonstrated for the first time the proliferation, maturation of chondrocytes, and matrix deposition on MBCP, suggesting the potential for such scaffold in tissue engineering via the endochondral bone formation mechanism..
"Intrauterine fetal constraint induces chondrocyte apoptosis and premature ossification of the cranial base"Smartt, James M Jr; Karmacharya, Jagajan; Gannon, Francis H; TEIXEIRA, CRISTINA; Mansfield, Kyle; Hunenko, Oksana; Shapiro, Irving M; Kirschner, Richard E
BACKGROUND: The spheno-occipital synchondrosis is an important growth center of the craniofacial skeleton and a primary site of malformation in syndromic forms of craniosynostosis. Clinical and laboratory investigations have demonstrated that premature closure of cranial vault sutures in nonsyndromic craniosynostosis is associated with characteristic alterations in cranial base morphology. However, a causal link between premature fusion of calvarial sutures and changes in the cranial base remains elusive. The purpose of these experiments was to test the hypothesis that intrauterine head constraint produces ultrastructural changes in the spheno-occipital synchondroses of fetal mice. METHODS: Fetal constraint was induced through uterine cerclage of six pregnant C57Bl/6 mice on the eighteenth day of gestation. Fetuses were harvested after growing to 24, 48, and 72 hours beyond the normal 20-day gestational period. Between six and nine fetuses were harvested at all time points in both treatment and control groups. The morphology and cell biology of the spheno-occipital synchondroses, in constrained fetuses and unconstrained controls, were examined using hematoxylin and eosin-stained sections. Chondrocyte apoptosis was examined using terminal deoxynucleotidyl transferase-mediated dUDP end-labeling assays and electron microscopy. RESULTS: In nonconstrained animals, the spheno-occipital synchondrosis demonstrated normal architecture and normal chondrocyte morphology at all time points. In contrast, intrauterine constraint resulted in a progressive disruption of the normal cellular architecture of the spheno-occipital synchondrosis over 72 hours, with premature ossification of the synchondrosis. Widespread chondrocyte apoptosis within the synchondrosial growth center was demonstrated by terminal deoxynucleotidyl transferase-mediated dUDP end-labeling assays and electron microscopy. CONCLUSION: These experiments confirm the ability of intrauterine constraint to induce changes in the morphology and cell biology of the cranial base in synostotic fetuses.