Effects of epidermal growth factor and keratinocyte growth factor on the growth of oropharyngeal keratinocytes in coculture with autologous fibroblasts in a three-dimensional matrix.

Tissue engineering of oropharyngeal mucosa is rendered complex by the fact that oropharyngeal keratinocytes are difficult to culture in the long term and do not grow well after several subcultivations. Three populations of oropharyngeal keratinocytes were isolated by a method based on different levels of beta(1)-integrin expression. In particular, keratinocytes were isolated between cell fractions that adhere rapidly on collagen-IV-coated culture dishes (RAC-IV) and populations that are less adherent (RAC-IV-D). The total fraction of both subpopulations served as a control (RAC-IV-T). The epidermal growth factor (EGF) and the keratinocyte growth factor (KGF) were examined with regard to their effects on the growth of the three populations. Growth curves of all three cell fractions grown with or without EGF were generated, and different concentrations of EGF and KGF were tested. EGF did not change any growth characteristics of the cells, with the exception of the speed of growth. Best growth was achieved with a physiologic EGF concentration of 0.15-1.5 ng/ml and a KGF concentration of 15 ng/ml. Finally, we cocultured oropharyngeal keratinocytes and their autologous fibroblasts in a three-dimensional matrix using Matrigeltrade mark. Oropharyngeal keratinocytes grown in coculture formed larger colonies than keratinocytes grown without fibroblasts. In conclusion, we were able to optimize the supplement of EGF and KGF in standard medium for the long-term culture of primary oropharyngeal keratinocytes. The use of Matrigel as a scaffold for three-dimensional cocultures of oropharyngeal keratinocytes and fibroblasts might signify a step forward in the development of a transplantable mucosa construct.

Three-dimensional composites manufactured with human mesenchymal cambial layer precursor cells as an alternative for sinus floor augmentation: an in vitro study.

Bone tissue engineering is a promising approach for treatment of defective and lost bone in the maxillofacial region. Creating functional tissue for load bearing bone reconstruction using biocompatible and biodegradable scaffolds seeded with living cells is of crucial importance. The aim of our study was to compare the effects of poly-lactic-co-glycolic acid (PLGA) and hydroxyapatite (HA) ceramic granulae on growth, differentiation, mineralization and gene expression of mandibular mesenchymal cambial layer precursor cells (MCLPCs) cultured onto tissue engineered three-dimensional (3-D) composites in vitro. These 3-D composites were cultivated in a rotating cultivation system under osteogenic differentiation conditions for a maximum period of 21 days. After 6 and 21 days, histological examination was performed; scanning electron microscopy (SEM), alkaline phosphatase (ALP) activity and levels of DNA were investigated. Expression of bone-specific genes osteocalcin, osteonectin, osteopontin, ALP, core binding factor alpha 1 and collagen type I were investigated by using a reverse transcription-polymerase chain reaction (RT-PCR) method. After 6 and 21 days of incubation an initiation of mineralization and the presence of newly formed bone at the surface of the composites were shown after evaluation of ALP activity, DNA content, SEM and histological staining. Expression of bone-specific genes confirmed the bone-like character of these composites and different effects of PLGA or HA granulae on the osteogenic differentiation of human MCLPCs in vitro. The results of this study support the concept that substrate signals significantly influence MCLPCs growth, differentiation, mineralization and gene expression in vitro, and that the use of these cells in the manufacturing of 3-D cell/HA composites is a promising approach for load bearing bone reconstruction in the maxillofacial region in vivo.

Mutation analysis of CLPTM 1 and PVRL 1 genes in patients with non-syndromic clefts of lip, alveolus and palate.

INTRODUCTION: Non-syndromic cleft lip with or without cleft palate (CL/P), is one of the most common birth defects, but its aetiology is largely unknown. The aim of this study was to determine the sequence changes of the Cleft Lip and Palate Transmembrane Protein 1 (CLPTM 1) and Poliovirus Receptor Related 1 (PVRL 1) genes in patients with non-syndromic complete clefts of lip, alveolus and palate and to correlate these findings with clinical features. PATIENTS AND METHODS: 25 patients were analysed (14 male and 11 female, aged 4-10 years) of European descent (9 patients with right, 9 with left and 7 patients with bilateral CLAP) and 25 controls, respectively. Exons 2-14 of the CLPTM1 and exons 1-6 of the PVRL1 gene were analysed by a direct sequencing method using DNA extracted from whole blood. RESULTS: A novel in frame Glu441-Gly442 ins Glu mutation of the PVRL 1 gene in combination with novel exon mutations Gly331Gly, Ala88Ala, Pro309Pro and intron change IVS7-10G/A of the CLPTM 1 gene were found in 9 patients. The Glu441-Gly442 ins Glu mutation and the intron change IVS7-10G/A were not detected in 25 controls. CONCLUSION: These results suggest that a simultaneous occurrence of PVRL1 and CLPTM 1 gene mutations in cleft patients does not correlate with the type of cleft (left, right, bilateral) or the gender of the patients. If a combination of the intron change IVS7-10G/A, exon changes Gly331Gly, Ala88Ala and Pro309Pro of the CLMPT 1 gene and Glu441-Gly442 ins Glu mutation of the PVRL 1 gene could be a genetic factor for non-syndromic clefts of the primary and the secondary palates, it is important to investigate more patients and controls.

In vitro growth and differentiation of osteoblast-like cells on hydroxyapatite ceramic granule calcified from red algae.

PURPOSE: The purpose of this study was to analyze the interaction between osteoblast-like cells isolated from mandibular bone and hydroxyapatite ceramic bone substitute obtained from calcified red algae to assess the growth and differentiation of adherent cells on this biomaterial. MATERIALS AND METHODS: The macroporous ceramic material C GRAFT/Algipore (The Clinician's Preference LLC, Golden, CO) is composed of 100% hydroxyapatite and possesses specific mechanical and physiochemical properties. Osteoblast-like cells were seeded on 200 mg of biomaterial and cultured for 6 and 21 days under osteogenic differentiation conditions. Specific alkaline phosphatase activity, DNA, and protein content of the proliferating cells were analyzed. The morphology of the cells in contact with the biomaterial was examined by scanning electron microscopy. The osteoblastic phenotype of the cells was confirmed by analysis of the expression of bone-specific genes (osteocalcin, osteopontin and collagen type I) by semi-quantitative reverse transcriptase polymerase chain reaction. RESULTS: The DNA and protein content increased over the culture period. Scanning electron microscopy showed cells spreading on the surface of the biomaterials, covering the macropores, and colonizing the depth of the particles. The analysis of the expression patterns of bone-related genes confirmed the osteoblastic phenotype of the cultured cells. CONCLUSION: The results of this study showed that hydroxyapatite ceramic bone substitute obtained from calcified red algae support the proliferation and differentiation of human osteoblast-like cells on its surface in vitro and might be suitable for use as scaffolds in tissue engineering strategies in vivo.

Analysis of cell-seeded 3-dimensional bone constructs manufactured in vitro with hydroxyapatite granules obtained from red algae.

PURPOSE: Bone tissue engineering is a promising approach for the treatment of defective or lost bone in the maxillofacial region. Biocompatible and biodegradable scaffolds seeded with living cells are used to create functional tissue for load-bearing bone reconstruction. The aim of this study was to manufacture cell-seeded 3-dimensional bone constructs based on hydroxyapatite ceramic granule calcified from red algae and mesenchymal cambial-layer precursor cells. The ability of these cells to grow on hydroxyapatite ceramic was quantitatively investigated to evaluate 3-dimensional bone constructs for their potential use in bone tissue engineering. MATERIALS AND METHODS: Mesenchymal cambial-layer precursor cells were isolated from mandibular periosteum biopsy samples of 3 patients. To manufacture 72 bone constructs, these cells and hydroxyapatite ceramic granules (C GRAFT/Algipore; Clinician's Preference LLC, Golden, CO) were cultivated under osteogenic differentiation conditions in a rotating wall vessel system. After 6 and 21 days, histologic examination and scanning electron microscopy were performed. The absolute DNA content, protein synthesis, and alkaline phosphatase activity were also quantified. The osteoblastic phenotype of the constructs was confirmed by the expression of bone-specific genes (osteocalcin, osteonectin, osteopontin, and core binding factor alpha1) using semiquantitative reverse transcription-polymerase chain reaction and Western blot analysis. RESULTS: Cells within the constructs showed good viability, which was evidenced by an increase in DNA content over the culture period. The decrease in alkaline phosphatase-specific activity could be an indicator of the maturation of cells and the induction of mineralization. The osteoblastic phenotype of the constructs was demonstrated on protein and at the RNA level over the entire culture period. CONCLUSION: We observed a positive effect of hydroxyapatite ceramic granules on mesenchymal cambial-layer precursor cell behavior in cell-seeded 3-dimensional bone constructs, indicating the potential applicability of C GRAFT/Algipore composites in bone tissue engineering.

Expression pattern of the chromosome 21 transcription factor Ets2 in cell-seeded three-dimensional bone constructs.

The ability to generate new bone for reconstructive surgery use is a major clinical need. Tissue engineering with osteoprogenitor cells isolated from the patient's periosteum and seeded into bioresorbable scaffolds offers a promising approach to the generation of skeletal tissue. To our knowledge, there is no description about the expression of Ets2 in tissue engineered "bone neotissue." The aim of our study was to manufacture cell-seeded three-dimensional bone constructs with human periosteal cells on poly (lactic-co-glycolic acid) polymer fleeces to describe the expression pattern of Ets2 and its target genes osteocalcin and osteopontin; expression analysis of type I collagen, core-binding factor-1, alkaline phosphatase, and osteonectin; the ability of matrix mineralization and ALP enzymatic activity showed the osteogenic character of the constructs. A significant correlation between the expression of Ets2 and osteopontin mRNA (r = -0.70; p < 0.05) could be shown. A 1.35-fold increase of Ets2 expression from days 1 to 9 was detected, followed by a slight decrease from days 11 to 15. Until the end of the culture period, the expression of Ets2 reached a comparable high level as detected on day 9. In contrast, the expression level of osteopontin mRNA reached a maximum at day 7, followed by a progressive 3.04-fold decrease until day 21. This study shows for the first time that Ets2 gene and its transcriptional target genes are expressed in tissue-engineered bone constructs. These findings have the potential to provide much-needed information about the role and function of Ets2 in human osteogenesis processes and creation of "bone neotissue."