Craniofacial Tissue Engineering.

The craniofacial complex is composed of fundamental components such as blood vessels and nerves, and also a variety of specialized tissues such as craniofacial bones, cartilages, muscles, ligaments, and the highly specialized and unique organs, the teeth. Together, these structures provide many functions including speech, mastication, and aesthetics of the craniofacial complex. Craniofacial defects not only influence the structure and function of the jaws and face, but may also result in deleterious psychosocial issues, emphasizing the need for rapid and effective, precise, and aesthetic reconstruction of craniofacial tissues. In a broad sense, craniofacial tissue reconstructions share many of the same issues as noncraniofacial tissue reconstructions. Therefore, many concepts and therapies for general tissue engineering can and have been used for craniofacial tissue regeneration. Still, repair of craniofacial defects presents unique challenges, mainly because of their complex and unique 3D geometry.

Bioelectric signalling via potassium channels: a mechanism for craniofacial dysmorphogenesis in KCNJ2-associated Andersen-Tawil Syndrome.

KEY POINTS: Xenopus laevis craniofacial development is a good system for the study of Andersen-Tawil Syndrome (ATS)-associated craniofacial anomalies (CFAs) because (1) Kcnj2 is expressed in the nascent face; (2) molecular-genetic and biophysical techniques are available for the study of ion-dependent signalling during craniofacial morphogenesis; (3) as in humans, expression of variant Kcnj2 forms in embryos causes a muscle phenotype; and (4) variant forms of Kcnj2 found in human patients, when injected into frog embryos, cause CFAs in the same cell lineages. Forced expression of WT or variant Kcnj2 changes the normal pattern of Vmem (resting potential) regionalization found in the ectoderm of neurulating embryos, and changes the normal pattern of expression of ten different genetic regulators of craniofacial development, including markers of cranial neural crest and of placodes. Expression of other potassium channels and two different light-activated channels, all of which have an effect on Vmem , causes CFAs like those induced by injection of Kcnj2 variants. In contrast, expression of Slc9A (NHE3), an electroneutral ion channel, and of GlyR, an inactive Cl(-) channel, do not cause CFAs, demonstrating that correct craniofacial development depends on a pattern of bioelectric states, not on ion- or channel-specific signalling. Using optogenetics to control both the location and the timing of ion flux in developing embryos, we show that affecting Vmem of the ectoderm and no other cell layers is sufficient to cause CFAs, but only during early neurula stages. Changes in Vmem induced late in neurulation do not affect craniofacial development. We interpret these data as strong evidence, consistent with our hypothesis, that ATS-associated CFAs are caused by the effect of variant Kcnj2 on the Vmem of ectodermal cells of the developing face. We predict that the critical time is early during neurulation, and the critical cells are the ectodermal cranial neural crest and placode lineages. This points to the potential utility of extant, ion flux-modifying drugs as treatments to prevent CFAs associated with channelopathies such as ATS. ABSTRACT: Variants in potassium channel KCNJ2 cause Andersen-Tawil Syndrome (ATS); the induced craniofacial anomalies (CFAs) are entirely unexplained. We show that KCNJ2 is expressed in Xenopus and mouse during the earliest stages of craniofacial development. Misexpression in Xenopus of KCNJ2 carrying ATS-associated mutations causes CFAs in the same structures affected in humans, changes the normal pattern of membrane voltage potential regionalization in the developing face and disrupts expression of important craniofacial patterning genes, revealing the endogenous control of craniofacial patterning by bioelectric cell states. By altering cells' resting potentials using other ion translocators, we show that a change in ectodermal voltage, not tied to a specific protein or ion, is sufficient to cause CFAs. By adapting optogenetics for use in non-neural cells in embryos, we show that developmentally patterned K(+) flux is required for correct regionalization of the resting potentials and for establishment of endogenous early gene expression domains in the anterior ectoderm, and that variants in KCNJ2 disrupt this regionalization, leading to the CFAs seen in ATS patients.

Tooth Tissue Engineering: The Importance of Blood Products as a Supplement in Tissue Culture Medium for Human Pulp Dental Stem Cells.

One of the goals in using cells for tissue engineering (TE) and cell therapy consists of optimizing the medium for cell culture. The present study compares three different blood product supplements for improved cell proliferation and protection against DNA damage in cultured human dental pulp stem cells for tooth TE applications. Human cells from dental pulp were first characterized as adult stem cells (ectomesenchymal mixed origin) by flow cytometry. Next, four different cell culture conditions were tested: I, supplement-free; II, supplemented with fetal bovine serum; III, allogeneic human serum; and IV, autologous human serum. Cultured cells were then characterized for cell proliferation, mineralized nodule formation, and colony-forming units (CFU) capability. After 28 days in culture, the comet assay was performed to assess possible damage in cellular DNA. Our results revealed that Protocol IV achieved higher cell proliferation than Protocol I (p = 0.0112). Protocols II and III resulted in higher cell proliferation than Protocol I, but no statistical differences were found relative to Protocol IV. The comet assay revealed less cell damage in cells cultured using Protocol IV as compared to Protocols II and III. The damage percentage observed on Protocol II was significantly higher than all other protocols. CFUs capability was highest using Protocol IV (p = 0.0018) and III, respectively, and the highest degree of mineralization was observed using Protocol IV as compared to Protocols II and III. Protocol IV resulted in significantly improved cell proliferation, and no cell damage was observed. These results demonstrate that human blood product supplements can be used as feasible supplements for culturing adult human dental stem cells.

Tooth tissue engineering: the influence of hydrophilic surface on nanocrystalline diamond films for human dental stem cells.

New techniques for tissue engineering (TE) are rapidly emerging. The basic concept of autologous TE is to isolate cells from small biopsy specimens, and to expand these cells in culture for subsequent seeding onto biodegradable scaffolds. Nanocrystalline diamond films have attracted the attention of researchers from a variety of different areas in recent years, due to their unique and exceptional properties. In this approach, human dental stem cells (hDSCs) were characterized by flow cytometry and grown on diamond films with hydrogen (H)-terminated and oxygen (O)-terminated surfaces for 28 days, and then removed by lysis and washing with distilled water. Energy dispersive spectroscopy analysis was performed, showing that the regions with O-terminated surfaces contained much higher levels of deposited calcium, oxygen, and phosphorus. These results suggest that the extracellular matrix was considerably more developed in the O-terminated regions, as compared with the H-terminated regions. In addition, optical microscopy of hDSCs cultured on the diamond substrate with H- and O-terminated surfaces, before washing with distilled water, showed preferential directions of the cells arrangement, where orthogonal lines suggest that the cells appeared to be following the O-terminated regions or hydrophilic surface. These findings suggest that O-terminated diamond surfaces prepared on biodegradable scaffolds can be useful for mineralized dental tissue formation.

Cytogenetic instability of dental pulp stem cell lines.

Human adult stem cells (hASCs) offer a potentially renewable source of cell types that are easily isolated and rapidly expanded for use in regenerative medicine and cell therapies without the complicating ethical problems that are associated with embryonic stem cells. However, the eventual therapeutic use of hASCs requires that these cells and their derivatives maintain their genomic stability. There is currently a lack of systematic studies that are aimed at characterising aberrant chromosomal changes in cultured ASCs over time. However, the presence of mosaicism and accumulation of karyotypic abnormalities within cultured cell subpopulations have been reported. To investigate cytogenetic integrity of cultured human dental stem cell (hDSC) lines, we analysed four expanded hDSC cultures using classical G banding and fluorescent in situ hybridisation (FISH) with X chromosome specific probe. Our preliminary results revealed that about 70% of the cells exhibited karyotypic abnormalities including polyploidy, aneuploidy and ring chromosomes. The heterogeneous spectrum of abnormalities indicates a high frequency of chromosomal mutations that continuously arise upon extended culture. These findings emphasise the need for the careful analysis of the cytogenetic stability of cultured hDSCs before they can be used in clinical therapies.

Tooth tissue engineering: optimal dental stem cell harvest based on tooth development.

Our long-term objective is to devise reliable methods to generate biological replacement teeth exhibiting the physical properties and functions of naturally formed human teeth. Previously, we demonstrated the successful use of tissue engineering approaches to generate small, bioengineered tooth crowns from harvested pig and rat postnatal dental stem cells (DSCs). To facilitate characterizations of human DSCs, we have developed a novel radiographic staging system to accurately correlate human third molar tooth developmental stage with anticipated harvested DSC yield. Our results demonstrated that DSC yields were higher in less developed teeth (Stages 1 and 2), and lower in more developed teeth (Stages 3, 4, and 5). The greatest cell yields and colony-forming units (CFUs) capability was obtained from Stages 1 and 2 tooth dental pulp. We conclude that radiographic developmental staging can be used to accurately assess the utility of harvested human teeth for future dental tissue engineering applications.

Zebrafish Wnt9b synteny and expression during first and second arch, heart, and pectoral fin bud morphogenesis.

Roles for Wnt9b in craniofacial development are indicated by the cleft lip mutant phenotype observed in the A/WySn mouse strain,(1) caused by a retrotransposon insertion mutation at the Wnt9b locus. Analyses of the zebrafish Wnt9b ortholog, wnt9b, were pursued to provide insight into early vertebrate craniofacial patterning events mediated by Wnt9b signaling. Zebrafish wnt9b cDNA clones were isolated and found to encode an open reading frame of 358 amino acids, with 68% amino acid identity to mouse Wnt9b and 70% amino acid identity to human WNT9B. Syntenic analyses demonstrated that wnt9b and wnt3 exist as a contiguous pair in amniote vertebrate species, and that these genes are separate in the zebrafish and Takifugu genomes. During the pharyngula period, a time of extensive growth and morphogenesis, zebrafish wnt9b exhibits discrete expression in dorsal and ventral first and second branchial arch tissues, the heart, and pectoral fin buds. These analyses suggest that in zebrafish, as in humans, wnt9b plays distinct roles in directing morphogenetic movements of developing branchial arch elements, and identify the zebrafish as a useful developmental model for the study of human craniofacial cleft lip and palate.