Formation of a new fibrous attachment to human dental roots

Summary This study was performed to improve currently employed in vitro models for the study of periodontal regeneration by using a porous filter upon which periodontal ligament cells were grown. Periodontal ligament cells were harvested and 0.3 mm root discs cut from three partially erupted and extracted third molar teeth of one patient. Experimental culturing was performed by seeding periodontal ligament cell suspensions on Puropor-200 filters supported by wire-mesh grids in Grobstein Petri dishes. The following day, an interdental space of 0.1 to 0.3 mm was created by gently placing two dental root discs upon the filter. Cultures were terminated after 42, 56, 112 and 124 days, and processed for light- and electron microscopy. Collagen fibril diameters were measured. Adjacent and often attached to large areas of cementum-lined root discs, a dense fiber fringe developed. This fiber fringe was not found on dentinlined root discs. Although less organized, older cultures demonstrated a similar disc-culture interface, which depended upon the presence or absence of original root cementum. Collagen fibrils of early cultures had a mean diameter of about 42 nm, while in older cultures the diameters ranged from 47 to 68 nm. It is concluded that the fibrous matrix attached to cementum-lined root discs somewhat resembles the initial stages of the formation of dental root cementum in vivo.     

In-vitro formation of a collagenous matrix upon previously diseased and experimentally treated cemental root surfaces

Summary A diseased and mechanically treated surface of root cementum is known, clinically, to favor periodontal regeneration. The present investigation was undertaken to test whether previously diseased and experimentally treated root surfaces can support the in-vitro formation of a new collagenous matrix. Three teeth extracted for advanced periodontitis were treated first with 5% sodium hypochlorite for 2 h to remove all organic material from the root surface. After the healthy, apical one third of the root was cut off, the roots were scaled with moderate pressure to remove visible calculus. Non-demineralized root discs were cut and placed on a co-culture of periodontal ligament- and alveolar bone-derived cells. After 7 weeks in culture, either one of two matrix types was found along the root surface. The most frequent matrix consisted of clusters of cells layered within densely aggregated collagen fibrils. The other, less frequent matrix consisted of loosely arranged collagen fibrils adjacent to the cemental surface. The findings support the notion that, in vitro, a collagenous matrix is formed in contact to diseased and experimentally treated root surfaces. However, the smooth, non-demineralized and scaled cemental surface does not appear to be a suitable substrate for interdigitation with newly produced collagen fibrils.     

Collagen processing, crosslinking, and fibril bundle assembly in matrix produced by fibroblasts in long-term cultures supplemented with ascorbic acid

Human foreskin fibroblasts were cultured for up to 6 weeks in medium supplemented with ascorbic acid. During this time, the cells produced an extensive new connective tissue matrix in which the accumulated collagen (mostly type I) amounted to about 0.25 mg/10(6) cells. The matrix was highly differentiated as shown by complete processing of procollagen to collagen alpha-chains and covalent crosslinking of the collagen. Alignment of collagen fibrils occurred as the fibrils were deposited between cells, and binding of adjacent fibrils to the cell surface appeared to hold the fibrils in register. Groups of aligned fibrils were subdivided into bundles by cell-surface folds. If beta-aminopropionitrile was added to the medium, collagen crosslinking was inhibited, but not collagen synthesis or fibril bundle organization. If ascorbic acid was omitted from the culture medium, the extensive new connective tissue matrix was not produced. Our results indicate that fibroblasts in long-term cultures supplemented with ascorbic acid produce a connective tissue matrix with many in vivo-like properties including supermolecular organization of collagen.     

Cultivation of human tenocytes in high-density culture

Limited supplies of tendon tissue for use in reconstructive surgery require development of phenotypically stable tenocytes cultivated in vitro. Tenocytes in monolayer culture display an unstable phenotype and tend to dedifferentiate, but those in three-dimensional culture may remain phenotypically and functionally differentiated. In this study we established a three-dimensional high-density culture system for cultivation of human tenocytes for tissue engineering. Human tenocytes were expanded in monolayer culture before transfer to high-density culture. The synthesis of major extracellular matrix proteins and the ultrastructural morphology of the three-dimensional cultures were investigated for up to 2 weeks by electron microscopy, immunohistochemistry, immunoblotting and quantitative, real-time PCR. Differentiated tenocytes were able to survive over a period of 14 days in high-density culture. During the culture period tenocytes exhibited a typical tenocyte morphology embedded in an extensive extracellular matrix containing cross-striated collagen type I fibrils and proteoglycans. Moreover, expression of the tendon-specific marker scleraxis underlined the tenocytic identity of these cells. Taken together, we conclude that the three-dimensional high-density cultures may be useful as a new approach for obtaining differentiated tenocytes for autologous tenocyte transplantation to support tendon and ligament healing and to investigate the effect of tendon-affecting agents on tendon in vitro.     

Epithelial-mesenchymal interactions: effects of a dental biomatrix on odontoblasts

The functional differentiation of odontoblasts requires specific interactions between these cells and the extracellular matrix. To further analyze these phenomena we studied the effects of a "dental papillae biomatrix" on isolated dental papillae cultured in vitro. The dental papillae biomatrix was extracted from EDTA-dissociated day-18 mouse dental papillae by homogenization, NaCl and enzymatic treatments, and deposited on Millipore filters. This biomatrix was studied by means of transmission electron microscopy and indirect immunofluorescence: it contained collagen fibrils, type IV collagen, fibronectin and laminin; cellular residues were also observed. The dental papillae were isolated by trypsin treatment of homologous tooth germs and cultured on uncoated (control) and coated filters. As shown by histological and cytological data, odontoblast-like cells never differentiated in control cultures. In presence of biomatrix and serum, polarized functional cells were observed. The functional state of these cells was enhanced by the addition of ascorbic acid to the culture media. Study of the incorporation of 3H-proline in cultured dental papillae and in macromolecules secreted into the culture media corroborated the morphological findings.     

Initiation of acellular extrinsic fiber cementum on human teeth

Summary The development of acellular extrinsic fiber cementum (AEFC) has never before been studied in human teeth. We have therefore examined the initiation of AEFC in the form of a collagenous fiber fringe and its attachment to the underlying dentinal matrix, in precisely selected, erupting human premolars with roots developed to 50%–60% of their final length. Freshly extracted teeth were prefixed in Karnovsky's fixative, decalcified in EDTA and subdivided into about 10 blocks each, cut from the mesial and distal root surfaces, vertical to and along the root axis. The blocks were postfixed in osmium tetroxide, embedded in Epon and cut for light- and electron-microscopic investigation. Starting at the advancing edge of the root, within a region extending about 1 mm coronal to this edge, fibroblast-like cells were seen closely covering the external root surface. Along the first 100 m from the root edge, these cells extended cytoplasmic processes and contacted the dentinal collagen fibrils. Between these cells and the dentinal matrix, new collagen fibrils and very short collagen fibers gradually developed. Within the second 100 m from the root edge, this resulted in the formation of a cell-fiber fringe network. Newly formed fibers of the fringe were directly attached to the non-mineralized matrix containing dentinal collagen fibrils and could be distinguished from the latter by differences in fibril orientation. During the process of dentin mineralization, the transitional zone between the fiber-fringe base and the dentinal matrix, i.e., the future dentino-cemental junction, also mineralized. It is suggested that this fiber fringe is the base of AEFC, which later increases in thickness by fiber extension and subsequent mineralization.Abbreviations AEFC acellular extrinsic fiber cementum - AIFC acellular intrinsic fiber cementum - CIFC cellular intrinsic fiber cementum - CMSC cellular mixed stratified cementum - ARE advancing root edge - CP cytoplasmic process - D dentin - DCJ dentinocemental junction - E enamel - EBL external basal lamina - EC epithelial cell - EDTA ethylene diaminetetraacetic acid - ERM epithelial rests of Malassez - FF fiber fringe - HRS Hertwig's epithelial root sheath - IBL internal basal lamina - MD mineralized dentin - NMD non-mineralized dentin - OB odontoblast - PD predentin - PL periodontal ligament     

SOMITE CHONDROGENESIS : A Structural Analysis

Light and electron microscopy are used in this study to compare chondrogenesis in cultured somites with vertebral chondrogenesis These studies have also characterized some of the effects of inducer tissues (notochord and spinal cord), and different nutrient media, on chondrogenesis in cultured somites Somites from stage 17 (54–60 h) chick embryos were cultured, with or without inducer tissues, and were fed nutrient medium containing either horse serum (HS) and embryo extract (EE), or fetal calf serum (FCS) and F12X Amino acid analyses were also utilized to determine the collagen content of vertebral body cartilage in which the fibrils are homogeneously thin (ca. 150 Å) and unbanded. These analyses provide strong evidence that the thin unbanded fibrils in embryonic cartilage matrix are collagen. These thin unbanded collagen fibrils, and prominent 200–800 Å protein polysaccharide granules, constitute the structured matrix components of both developing vertebral cartilage and the cartilage formed in cultured somites Similar matrix components accumulate around the inducer tissues notochord and spinal cord. These matrix components are structurally distinct from those in embryonic fibrous tissue The synthesis of matrix by the inducer tissues is associated with the inductive interaction of these tissues with somitic mesenchyme. Due to the deleterious effects of tissue isolation and culture procedures many cells die in somitic mesenchyme during the first 24 h in culture. In spite of this cell death, chondrogenic areas are recognized after 12 h in induced cultures, and through the first 2 days in all cultures there are larger accumulations of structured matrix than are present in equivalently aged somitic mesenchyme in vivo. Surviving chondrogenic areas develop into nodules of hyaline cartilage in all induced cultures, and in most non-induced cultures fed medium containing FCS and F12X There is more cell death, less matrix accumulation, and less cartilage formed in cultures fed medium containing HS and EE. The inducer tissues, as well as nutrient medium containing FCS and F12X, facilitate cell survival, the synthesis and accumulation of cartilage matrix, and the formation of cartilage nodules in cultured somites.     

Cellular remodelling of individual collagen fibrils visualized by time-lapse AFM

The extracellular matrix in tissues such as bone, tendon and cornea contains ordered, parallel arrays of collagen type I fibrils. Cells embedded in these matrices frequently co-align with the collagen fibrils, suggesting that ordered fibrils provide structural or signalling cues for cell polarization. To study mechanisms of matrix-induced cell alignment, we used nanoscopically defined two-dimensional matrices assembled of highly aligned collagen type I fibrils. On these matrices, different cell lines expressing integrin alpha(2)beta(1) polarized strongly in the fibril direction. In contrast, alpha(2)beta(1)-deficient cells adhered but polarized less well, suggesting a role of integrin alpha(2)beta(1) in the alignment process. Time-lapse atomic force microscopy (AFM) demonstrated that during alignment cells deform the matrix by reorienting individual collagen fibrils. Cells deformed the collagen matrix asymmetrically, revealing an anisotropy in matrix rigidity. When matrix rigidity was rendered uniform by chemical cross-linking or when the matrix was formed from collagen fibrils of reduced tensile strength, cell polarization was prevented. This suggested that both the high tensile strength and pliability of collagen fibrils contribute to the anisotropic rigidity of the matrix, leading to directional cellular traction and cell polarization. During alignment, cellular protrusions contacted the collagen matrix from below and above. This complex entanglement of cellular protrusions and collagen fibrils may further promote cell alignment by maximizing cellular traction.     

Structure of the periodontal ligament during tooth eruption in the dog: descriptive aspects

Serially stained uncalcified sections of young dog mandibles were examined to study the structure of the periodontal ligament of the erupting first right molar. The periodontal ligament around tooth crown presents three zones. The first, near the dental follicle, is a tick layer of parallel collagen bundles with numerous flattened fibroblasts. The second, intermediate, contains a blood vessels network, particularly veins and capillaries. The third, outer, is occupied by a continuous layer of osteoclasts and osteoblasts. Also the periodontal ligament around the tooth presents three layers, the outer and the intermediate rich of cells more than the inner. Particularly, the outer layer shows numerous osteoblasts surrounding the developing trabeculae of the alveolar bone and the collagen fiber bundles of the periodontal ligament. These penetrate into the trabeculae and appear similar to the osteoid layer. These results indicate that the alveolar bone increases by ossification of the connective tissue of the periodontal ligament.     

Synthesis of cartilage matrix by mammalian chondrocytes in vitro. III. Effects of ascorbate

Chondrocytes isolated from bovine articular cartilage were plated at high density and grown in the presence or absence of ascorbate. Collagen and proteoglycans, the major matrix macromolecules synthesized by these cells, were isolated at times during the course of the culture period and characterized. In both control and ascorbate-treated cultures, type II collagen and cartilage proteoglycans accumulated in the cell-associated matrix. Control cells secreted proteoglycans and type II collagen into the medium, whereas with time in culture, ascorbate-treated cells secreted an increasing proportion of types I and III collagens into the medium. The ascorbate-treated cells did not incorporate type I collagen into the cell-associated matrix, but continued to accumulate type II collagen in this compartment. Upon removal of ascorbate, the cells ceased to synthesize type I collagen. Morphological examination of ascorbate-treated and control chondrocyte culture revealed that both collagen and proteoglycans were deposited into the extracellular matrix. The ascorbate-treated cells accumulated a more extensive matrix that was rich in collagen fibrils and ruthenium red-positive proteoglycans. This study demonstrated that although ascorbate facilitates the formation of an extracellular matrix in chondrocyte cultures, it can also cause a reversible alteration in the phenotypic expression of those cells in vitro.     

Phenotypic analysis of bovine chondrocytes cultured in 3D collagen sponges: effect of serum substitutes

Repair of damaged cartilage usually requires replacement tissue or substitute material. Tissue engineering is a promising means to produce replacement cartilage from autologous or allogeneic cell sources. Scaffolds provide a three-dimensional (3D) structure that is essential for chondrocyte function and synthesis of cartilage-specific matrix proteins (collagen type II, aggrecan) and sulfated proteoglycans. In this study, we assessed porous, 3D collagen sponges for in vitro engineering of cartilage in both standard and serum-free culture conditions. Bovine articular chondrocytes (bACs) cultured in 3D sponges accumulated and maintained cartilage matrix over 4 weeks, as assessed by quantitative measures of matrix content, synthesis, and gene expression. Chondrogenesis by bACs cultured with Nutridoma as a serum replacement was equivalent or better than control cultures in serum. In contrast, chondrogenesis in insulin-transferrin-selenium (ITS⁺³) serum replacement cultures was poor, apparently due to decreased cell survival. These data indicate that porous 3D collagen sponges maintain chondrocyte viability, shape, and synthetic activity by providing an environment favorable for high-density chondrogenesis. With quantitative assays for cartilage-specific gene expression and biochemical measures of chondrogenesis in these studies, we conclude that the collagen sponges have potential as a scaffold for cartilage tissue engineering.     

The effect of ascorbate on the synthesis of minor (non-interstitial) collagens by cultured bovine aortic smooth muscle cells

Ultrastructural and biochemical studies were carried out on bovine aortic smooth muscle cells cultured in the presence or absence of ascorbate. In its absence, electron microscopic examination of cultures revealed that the extracellular components consisted primarily of microfibrils. Morphologically identifiable collagen fibrils were only observed in the matrix upon ascorbate supplementation. Smooth muscle cells grown in ascorbate-free media synthesized large amounts of type VI collagen. The identity of the latter was confirmed by ion exchange chromatography, slab gel electrophoresis, and amino acid analysis. Addition of ascorbate resulted in a stimulation of type I collagen production, levels of the type III remained constant, and types V and VI were decreased. Since, in the absence of ascorbate, smooth muscle cells are known to synthesize predominantly elastin, the present data support the contention that the type VI collagen and the microfibrillar component of elastic tissue are either identical or similar.     

Survival of rat functional dental pulp cells in vascularized tissue engineering chambers

Regenerative endodontics aims to preserve, repair or regenerate the dental pulp tissue. Dental pulp stem cells, have a potential use in dental tissue generation. However, specific requirements to drive the dental tissue generation are still obscured. We established an in vivo model for studying the survival of dental pulp cells (DPC) and their potential to generate dental pulp tissue. DPC were mixed with collagen scaffold with or without slow release bone morphogenic protein 4 (BMP-4) and fibroblast growth factor 2 (FGF2). The cell suspension was transplanted into a vascularized tissue engineering chamber in the rat groin. Tissue constructs were harvested after 2, 4, 6, and 8 weeks and processed for histomorphological and immunohistochemical analysis. After 2 weeks newly formed tissue with new blood vessel formation were observed inside the chamber. DPC were found around dentin, particularly around the vascular pedicle and also close to the gelatin microspheres. Cell survival, was confirmed up to 8 weeks after transplantation. Dentin Sialophosphoprotein (DSPP) positive matrix production was detected in the chamber, indicating functionality of dental pulp progenitor cells. This study demonstrates the potential of our tissue engineering model to study rat dental pulp cells and their behavior in dental pulp regeneration, for future development of an alternative treatment using these techniques.     

Localization of calcium and phosphorus in early predentin-matrix components by electron spectroscopic imaging (ESI)-analysis in rat molars

Summary The subcellular distribution of the inorganic elements calcium (Ca) and phosphorus (P) was studied in the first-formed dentin matrix during initial mineralization in neonatal rat molars. This most peripheral matrix region is comprised of a proteoglycan-rich ground substance, interwoven by a collagenous network, matrix vesicles, aperiodic fibrils derived from the dental basal lamina, and apical odontoblastic cell processes. All matrix components may possibly serve as templets for mineral deposition during initial calcification of first-formed mantle dentin and predentin. By means of the very sensitive ESI-analysis we studied the subcellular localization of Ca and P and their possible association with distinct organic extracellular matrix components and odontoblasts. Ca-signals were found in the ground substance, at striated collagen fibrils and plasma membranes of odontoblasts in the cuspal early matrix region, but occurred only sparsely in the ground substance of the more distal matrix region where odontoblast processes attach to aperiodic fibrils of the dental basal lamina. Ca was generally absent in matrix vesicles. In contrast, P-signals were found in matrix vesicles, at aperiodic fibrils and at the plasma membranes of odontoblasts. Ca and P co-localized at striated collagen fibrils (type I or II). These results suggest that striated collagen fibrils might serve as primary deposition sites for calcium phosphate during early biological calcification of organic extracellular macromolecules.     

Directing the differentiation of human dental follicle cells into cementoblasts and/or osteoblasts by a combination of HERS and pulp cells

It is known that the dental follicle (DF) consists of progenitor cells that give rise to the cementum, periodontal ligament, and alveolar bone; but little information is available about the regulation of DF cell differentiation into either cementogenic or osteogenic cell lineages for the regeneration of diseased periodontal tissue. Here, we investigated the roles of DF, Hertwig’s epithelial root sheath (HERS), and pulp cells in the cementum and during alveolar bone formation. We cultured these cells; transplanted them alone or in combination into immunocompromised mice; and observed their effects at 6 and 12 weeks. Histological and immunohistochemical results revealed that DF cells formed cementum-like tissues with immunoreactivity to cementum-derived attached protein, bone sialoprotein, type I collagen, and alkaline phosphatase. In addition, HERS cells played a role in the induction and maturation of cementum-like tissues formed by DF cells. In contrast, implants of DF cells in the presence of pulp cells led to the formation of bone-like tissues. Interestingly, in the presence of both HERS and pulp cells, DF cells formed both cementum-like and bone-like tissues. We demonstrated that while HERS cells are able to induce DF cell differentiation into cementoblasts and promote cementum formation, pulp cells could direct DF cell differentiation into osteoblasts and enhance alveolar bone formation. These results suggest that the combined use of DF, HERS, and pulp cells could direct DF cell differentiation into cementoblasts and/or osteoblasts in vivo, thus providing a novel strategy for the successful repair and regeneration of diseased periodontal tissue.     

Morphology, differentiation and matrix production of liver cells in organoid cultures (high density cultures) of fetal rat livers.

The aim of this study was to demonstrate the morphology and matrix synthesis of embryonic rat liver cells (day 18 of gestation) in organoid cultures (high density cultures) with electron microscopic and immunomorphological techniques. For this purpose the cells of embryonic rat livers were isolated enzymatically and grown in an organoid culture (high density culture) for 3 weeks in a Trowell system. During the first 48 h a sorting-out process took place, i.e. liver and blood-forming cells met to form aggregates. In between mesenchymal cells were seen. Vessel-like cavities developed. Electron microscopic inspection of the hepatocytes did not reveal any lesions of the cell organelles after 14 days in culture. As late as after a 3-week culture period mitochondrial swellings and an increased number of autophagic vacuoles were observed. A rim of collagenous fibrils or fibrillar bundles and granular matrix structures was perceptible as early as after 7 days in culture. Immunofluorescence microscopic techniques revealed collagen types III, IV and VI as well as laminin, nidogen, heparansulfate-proteoglycan and fibronectin in these areas. Thus, the composition of the matrix in this culture system corresponds (apart from the absence of collagen type I) to the embryonic situation. Therefore, the organoid culture appears to be an appropriate technique to study the behaviour of hepatocytes in vitro. It is especially suited to demonstrate the formation of matrix components in liver cells and their extracellular occurrence.     

Cartilage induction in vitro : Ultrastructural studies

When 19-day fetal rat triceps muscle was cultured for 7 to 14 days upon decalcified, sequentially extracted adult rat bone, cartilage formed within clefts and vascular spaces of the decalcified bone. The bone substrata were prepared by extracting tibias and femurs of Sprague-Dawley rats with 1:1 chloroform:methanol, 0.6 N HCl, 2 M CaCl₂, 0.6 M EDTA, 8 M LiCl, and H₂O at 56°C. The culture medium used was CMRL 1066 with 15% newborn calf serum. During cultivation, fibroblastic mesenchymal cells migrated out of muscle and into bone crevices where they secreted a cartilaginous matrix composed of thin, randomly dispersed collagen fibrils and proteoglycan granules. The latter are characteristic for cartilage matrix. Extracted bone matrix contained mature collagen fibrils, some of which retained their typical 640-Å banding. Other collagen fibrils were partially disaggregated and expanded to reveal component 50-Å-thick, beaded micro fibrils. Such an expansion of collagen fibrils is known to result from exposure to proteoglycan solvents such as 2 M CaCl₂. The decalcified bone matrix contained many residual devitalized cells and cell fragments which often were seen in close proximity to chondrifying mesenchymal cells. This finding indicates the possibility that residual cellular material could play a role in stimulating cartilage development.     

Formation of Intercellular Collagen Matrix by Cultured Liver Epithelial Cells and Loss of its Ability in Hepatocarcinogenesis in vitro

Epithelial cells of normal rat liver origin (strain RL34) synthesized the α1 peptide of type I collagen. In nononcogenic cultures (RL34 and RL34EC) and a marginally oncogenic culture (RL34HII), the peptide was continuously secreted from the proliferating cells. Part of the soluble peptide was incorporated into the intercellular matrix of contact-inhibited cells after confluency, while the remainder was degraded. The intercellular matrix contained characteristic collagen fibrils which were argyrophilic and revealed a 64 nm axial periodicity. Epithelial cells of an oncogenic culture (RL34HT) secreted procollagen into the medium continuously throughout their proliferative phases and were unable to accumulate collagen fibrils in the intercellular matrix. The depletion of collagen accumulation in the hepatocarcinoma cell culture was ascribed to lack of the binding of native collagen molecules to the cell membrane and the persistence of high proteolytic activity on the cell surface.     

Different substrates influence the expression of intermediate filaments and the deposition of basement membrane proteins

Summary A primary culture of serous cystadenocarcinoma of the ovary was used to study the expression of intermediate filament proteins and the deposition of basal lamina proteins. It was found that cells grown on type I and IV collagens or in collagen gels failed to express vimentin, which was readily demonstrable in cultures of the same cells grown on plastic or glass. Furthermore cells grown in collagen gels formed colonies demonstrating a cystic architecture Unlike what is commonly observed on glass or plastic where laminin and fibronectin are deposited as disorganized fibrils in the extracellular space, in or on collagen these proteins appear solely at the interface between the epithelial cells and matrix. The results suggest that the extracellular matrix influences the cytoskeletal organization of the intermediate filaments and determines cell polarity. They confirm that collagen substrates permit epithelial cell cultures to progress toward a more differentiated state. Supported by grants from the Italian Assciation for Cancer Research (AIRC).