Periosteal Sheets Research Articles

Non-destructive, spectrophotometric analysis of the thickness of the cell-multilayered periosteal sheet

BackgroundAutologous tissue-engineered periosteal sheets, which have been clinically applied for periodontal regeneration, sinus lift, and alveolar ridge augmentation, are enriched with osteoblast precursor cells and the abundant deposition of collagen type I in the extracellular spaces. Their quality is inspected prior to clinical use; however, most criteria cannot be evaluated without sacrificing samples. To reduce such losses, we developed a non-destructive optical method that can quantitatively evaluate the thickness of the periosteal sheet.MethodsDispersed periosteal cells were inoculated into small pieces of collagen sponge (Terudermis®) and plated into 60-mm dishes for further explant culture using a conventional medium and a stem-cell culture medium. The thickness of periosteal sheets was evaluated using inverted microscopic, histological, labeling (CellVue®)-based imaging and spectrophotometric (Spectro-1®) methods.ResultsThe three-dimensional growth of periosteal sheets did not necessarily correlate with two-dimensional growth. The periosteal sheet prepared with the stem-cell medium formed cell multilayers, a phenomenon that could be observed qualitatively by inverted microscopy. The spectrophotometric analysis enabled the quantitative evaluation of the thickness of the cell multilayer without sacrificing the samples processed for scheduled cell therapy.ConclusionsThe growth of periosteal sheets is influenced by several major factors, including the basic quality of the individual original periosteal tissue segments, the technical expertise of doctors and operators involved in tissue harvesting and processing, and culture conditions. This newly developed spectrophotometric analysis can quantify the thickness of cell-multilayered periosteal sheets for quality assurance in a non-destructive manner, thereby contributing to better bone augmentation prior to implant therapy.

Osteoclastogenic Potential of Tissue-Engineered Periosteal Sheet: Effects of Culture Media on the Ability to Recruit Osteoclast Precursors.

Cell culture media influence the characteristics of human osteogenic periosteal sheets. We have previously found that a stem cell medium facilitates growth and collagen matrix formation in vitro and osteogenesis in vivo. However, it has not yet been demonstrated which culture medium is superior for osteoclastogenesis, a prerequisite for reconstruction of normal bone metabolic basis. To address this question, we compared chemotaxis and osteoclastogenesis in tissue-engineered periosteal sheets (TPSs) prepared with two types of culture media. Periosteal tissues obtained from adult volunteers were expanded with the conventional Medium 199 or with the stem cell medium, MesenPRO. Hematopoietic enhanced-green-fluorescent-protein (EGFP)-nude mice were prepared by γ-irradiation of Balb/c nu/nu mice and subsequent transplantation of bone marrow cells from CAG-EGFP C57BL/6 mice. TPSs were implanted subcutaneously into the chimeric mice and retrieved after intervals for immunohistopathological examination. EGFP+ cells were similarly recruited to the implantation site in both the TPSs prepared, whereas the distribution of CD11b+ cells was significantly lower in the TPS prepared with the stem cell medium. Instead, osteoclastogenesis was higher in the TPS prepared with the stem cell medium than in the one prepared with the conventional medium. These findings suggest that the stem cell medium is preferable for the preparation of more functional TPSs.

Platelet-Rich Fibrin Extract: A Promising Fetal Bovine Serum Alternative in Explant Cultures of Human Periosteal Sheets for Regenerative Therapy.

In 2004, we developed autologous periosteal sheets for the treatment of periodontal bone defects. This regenerative therapy has successfully regenerated periodontal bone and augmented alveolar ridge for implant placement. However, the necessity for 6-week culture is a limitation. Here, we examined the applicability of a human platelet-rich fibrin extract (PRFext) as an alternative to fetal bovine serum (FBS) for the explant culture of periosteal sheets in a novel culture medium (MSC-PCM) originally developed for maintaining mesenchymal stem cells. Small periosteum tissue segments were expanded in MSC-PCM + 2% PRFext for 4 weeks, and the resulting periosteal sheets were compared with those prepared by the conventional method using Medium199 + 10% FBS for their growth rate, cell multilayer formation, alkaline phosphatase (ALP) activity, and surface antigen expression (CD73, CD90, and CD105). Periosteal sheets grew faster in the novel culture medium than in the conventional medium. However, assessment of cell shape and ALP activity revealed that the periosteal cells growing in the novel medium were relatively immature. These findings suggest that the novel culture medium featuring PRFext offers advantages by shortening the culture period and excluding possible risks associated with xeno-factors without negatively altering the activity of periosteal sheets.

Synergistic effects of the combined use of human-cultured periosteal sheets and platelet-rich fibrin on bone regeneration: An animal study.

A human‐cultured alveolar bone‐derived periosteal (hCP) sheet is an osteogenic grafting material used clinically in periodontal regenerative therapy, while platelet‐rich fibrin (PRF), a platelet concentrate with fibrin clot, is considered to augment the wound healing process. Therefore, whether the combined use of hCP‐PRF complex could facilitate bone regeneration synergistically was evaluated in animal models. Human periosteal segments (1 × 1 mm) were cultured initially on plastic dishes and formed an hCP sheet. The hCP sheet was implanted with freshly prepared human PRF into subcutaneous tissue (hCP: n = 4, hCP + PRF: n = 4) and 4 mm diameter calvarial bone defect models (hCP: n = 4, hCP + PRF: n = 4, control [defect‐only]: n = 4) that prepared in nude mice. At 4 weeks postimplantation, new bone formation was evaluated by using μCT. Cell growth and neovascularization were evaluated by histochemical and immunohistological methods. In the subcutaneous tissue, mineral deposit formation, collagen deposition, and number of vessels were higher in the hCP + PRF group than in the hCP alone group. In the calvarial defect models, new bone formation was significantly higher in the hCP + PRF group than in the hCP alone group and defect‐only control group. The numbers of vessels and PCNA‐positive cells in calvarial defects were also increased in the hCP + PRF group more than in the hCP alone group. Platelet‐rich fibrin preparations support the proliferation and the growth of periosteal cells to form well‐combined active biological materials. Platelet‐rich fibrin also stimulates the local angiogenesis in the implantation site. Therefore, the combined use of hCP and PRF could be clinically applicable in bone regeneration therapy.

Real-time quantitative polymerase chain reaction and flow cytometric analyses of cell adhesion molecules expressed in human cell–multilayered periosteal sheets in vitro

Background aimsCultured human periosteal sheets more effectively function as an osteogenic grafting material at implantation sites than do dispersed periosteal cells. Because adherent cell growth and differentiation are regulated by cell-cell and cell–extracellular matrix contacts, we hypothesized that this advantage is a result of the unique cell adhesion pattern formed by their multiple cell layers and abundant extracellular matrix. To test this hypothesis, we prepared three distinct forms of periosteal cell cultures: three-dimensional cell-multilayered periosteal sheets, two-dimensional dispersed cell cultures, and three-dimensional hybrid mock-ups of cells dispersed onto collagen sponges. MethodsPeriosteal cells were obtained from human alveolar bone. Cell adhesion and extracellular matrix molecules were quantitatively determined at the messenger RNA and protein levels by means of real-time quantitative polymerase chain reaction and flow cytometry, respectively. ResultsReal-time quantitative polymerase chain reaction analysis demonstrated that regardless of culture media α1 integrin, vascular cell adhesion molecule-1, fibronectin and collagen type 1 were substantially upregulated, whereas CD44 was strongly downregulated in periosteal sheets compared with dispersed cell monolayers. With increased thickness, stem cell medium upregulated several integrins (β1, α1 and α4), CD146, vascular cell adhesion molecule-1, fibronectin and collagen type 1 in the periosteal sheets. Flow cytometric analysis revealed that the active configuration of β1 integrin was substantially downregulated in the stem cell medium–expanded cell cultures. The cell adhesion pattern found in the mock-up cultures was almost identical to that of genuine periosteal sheets. ConclusionsIntegrin α1β1 and CD44 function as the main cell adhesion molecule in highly cell-multilayered periosteal sheets and dispersed cells, respectively. This difference may account for the more potent osteogenic activity shown by the thicker periosteal sheets.

Biological and biomechanical characterization of highly self-multilayered human periosteal sheets as an osteogenic grafting material

Biological and biomechanical characterization of highly self-multilayered human periosteal sheets as an osteogenic grafting material

Biomechanical evaluation by AFM of cultured human cell-multilayered periosteal sheets

We previously demonstrated that thicker periosteal sheets with enhanced cell layering maintain their component cells at relatively immature stages of differentiation but express a high in vivo osteogenic potential. As it has been recently proposed that stiff scaffolds provide a mechanical cue to various cell types that promotes differentiation, we postulated that the maintenance of immature cells in our periosteal sheets is due to the mechanical stiffness of the multilayered-cell architecture. To demonstrate the biomechanical characteristics of our periosteal sheets, we have determined their stiffnesses with atomic force microscopy (AFM) and evaluated the expression of extracellular matrix (ECM) components specifically by both immunocytochemistry and a complementary DNA microarray technology. Compared to osteoblastic Saos2 cells, the cytoskeletal fibers were developed more in the periosteal cells, but the periosteal cells in monolayer culture developed before either the cells in the peripheral or central regions of the periosteal sheets developed. However, the nanoindentation by AFM distinguished the central region from the peripheral region. The peak stiffness values of cells were ordered as follows: tissue culture polystyrene (1.66GPa)≫dispersed (9.99kPa)>central region (5.20kPa)>peripheral regions (3.67kPa). Similarly, the degree of development of α-smooth muscle actin (αSMA) filaments within cells was dispersed>central region>peripheral region. In conjunction with the abundantly deposited ECM in the periosteal sheets, these findings suggest that the order of cell stiffness may depend on the integration of the stiffness of individual ECM components and the extent of cytoskeletal fiber formation. Because recently published data have demonstrated that the optimal stiffness for osteogenic differentiation is 25–40kPa, it is plausible that the periosteal cells residing in the less-stiff multilayer regions could be maintained at relatively immature stages under the control of the stem-cell medium in vitro but start differentiating when exposed to the proper stiffness upon release from the culture conditions at the implantation site.

Application of stem-cell media to explant culture of human periosteum: An optimal approach for preparing osteogenic cell material

As part of our clinical tests on bone regeneration using cultured periosteal sheets, here, we prepared cultured periosteal sheets in two types of stem-cell culture media, STK1 and STK3. Human periosteum was expanded either in 1% human serum–supplemented STK1 for 28 days, in 1% human serum–supplemented STK1 for 14 days followed by 1% human serum–supplemented STK3 for 14 days (1% human serum–supplemented STK1+3), or in 10% fetal bovine serum–supplemented Medium 199 for 28 days (control). Cultured periosteal sheet diameter and DNA content were significantly higher, and the multilayer structure was prominent in 1% human serum–supplemented STK1 and 1% human serum–supplemented STK1+3. The messenger RNA of osteoblastic markers was significantly upregulated in 1% human serum–supplemented STK1+3. Osteopontin-immunopositive staining and mineralization were evident across a wide area of the cultured periosteal sheet in 1% human serum–supplemented STK1+3. Subcutaneous implantation in nude mice following expansion in 1% human serum–supplemented STK1+3 produced the highest cultured periosteal sheet osteogenic activity. Expansion in 1% human serum–supplemented STK1+3 successfully induced cultured periosteal sheet growth while retaining osteogenic potential, and subsequent osteoblastic induction promoted the production of homogeneous cell material.

Microporous membranes of PLLA/PCL blends for periosteal tissue scaffold

Flexible microporous scaffold membranes of poly(l-lactic acid) (PLLA)/poly(ε-caprolactone) (PCL) blend were developed for bone regeneration. The new membranes overcome the fragility problems of PLLA membranes. When the PCL content in the blend was increased to 50%, the mechanical property for elongation was significantly improved without sacrificing the pores on the membrane surface that help tissue segments adhere the membrane. However, further increases in PCL content reduced the surface pores. In addition, spontaneous cell invasion and formation of cell-multilayers were observed in the membrane of PLLA/PCL blend (50:50). These results indicate that the porous membrane of PLLA/PCL blend (50:50) is useful for the preparation of periosteal sheets in tissue engineering.

Tissue culture of human alveolar periosteal sheets using a stem-cell culture medium (MesenPRO-RS™): In vitro expansion of CD146-positive cells and concomitant upregulation of osteogenic potential in vivo

We have previously demonstrated that multilayered periosteal sheets prepared from the explant culture of alveolar periosteum serve as a promising osteogenic grafting material in periodontal tissue regeneration. For the preparation of more potent periosteal sheets, we examined the applicability of stem-cell culture media. Compared to the control medium (Medium 199+10% FBS), periosteal sheets expanded with MesenPRO-RS™ medium exhibited these features: Cells grew three-dimensionally and deposited collagen in the extracellular spaces to form thicker multilayers of cells. Chondrocytic markers were not significantly upregulated. Contractile force was generated in proportion with the increased thickness of the periosteal sheets and the formation of cytoplasmic α-smooth muscle actin fibers. However, myofibroblastic markers were not significantly upregulated. The surface marker CD146 was substantially upregulated, while both CD73 and CD105 were downregulated. Alkaline phosphatase, a representative osteoblastic marker, was not upregulated by osteogenic induction. However, these expanded periosteal sheets exhibited substantially stronger osteogenic differentiation when implanted in nude mice. Therefore, despite our reservations, MesenPRO medium effectively expanded the cells contained in periosteal sheets to promote the formation of thicker multilayers of cells in vitro, and these enhanced periosteal sheets expressed increased osteogenic potential at implantation sites in vivo. In conjunction with data indicating that CD146-positive cells were notably expanded and the recently proposed concept that CD146 is a marker for osteogenic progenitor cells found in the bone marrow stroma, our findings suggest that MesenPRO medium improves the preparation of highly osteogenic periosteal sheets suitable for clinical application largely through the induction of CD146-positive cells.

A short-term preservation of human cultured periosteal sheets, osteogenic grafting materials, using a commercial preservation solution containing epigallocatechin-3-gallate (Theliokeep(®)) under hypothermic conditions.

In the past decade, it has increasingly been reported that epigallocatechin-3-gallate (EGCG), a major catechin derivative extracted from Green tea, has various bioactivities, including a cell-protective action on mammalian cells and tissues. In this study, we have tested a commercial preservation solution containing EGCG (Theliokeep(®)) in both two- and three-dimensional cultures of human periosteal sheets, which have been used as an osteogenic grafting material for periodontal regenerative therapy. When periosteal sheets were 3D-cultured on collagen mesh, cell viability was maintained for 2 days using the hypothermic EGCG preservation solution. Replenishment of EGCG solution with 2-day intervals prevented the time-dependent decline in cell viability at 3 days and later. As observed in nonpreserved control cultures, most cells were positive for proliferating cell-nuclear antigen (PCNA) in the cultures preserved at 4°C in the EGCG solution, whereas PCNA-negative cells were increased in the cultures preserved at 4°C in the MesenPRO medium. In periosteal sheets 2D-cultured in plastic dishes, the EGCG solution occasionally was associated with vacuole formation in the cytoplasm, but cells could again expand in the culture medium at 37°C. As observed in the nonpreserved periosteal sheets control, the osteogenic induction upregulated alkaline phosphatase in those cells and tissues preserved in the EGCG solution. The EGCG solution protected cells from the cold shock-induced membrane phospholipid peroxidation. Our data suggest that the EGCG solution acts as an antioxidant to protect periosteal cells from cold shock and preserves cells under chilled conditions. The limited period of preservation time could be expanded by repeating replenishment of the EGCG solution or by optimizing the formula to be more favorable for human periosteal sheets without sacrificing cell viability. This methodology of preserving human cultured periosteal sheets with EGCG would be expected to support and spread the clinical use of regenerative therapy with autologous periosteal sheets.

An osteogenic grafting complex combining human periosteal sheets with a porous poly(l-lactic acid) membrane scaffold: Biocompatibility, biodegradability, and cell fate in vivo

In this in vitro study, novel porous poly(l-lactic acid) membranes were developed to improve periosteal sheets by promoting initial adhesion of periosteal tissue segments and stimulating the formation of a viable multilayered cellular sheet. The biocompatibility, biodegradability, and osteogenicity were evaluated using human periosteal tissue segments cultured on porous poly(l-lactic acid) membranes; the periosteal sheets were osteogen induced and were then implanted in the dorsal subcutaneous tissue of nude mice. In vivo, the membrane degraded into clusters of membrane particles separated by wide cracks; fibroblastic cells invaded along with small blood vessels from the surrounding mouse connective tissue. In osteoinduced periosteal sheets, the membrane clusters were surrounded by numerous capillaries and a number of tartrate-resistant acid phosphatase–positive, multinucleated cells. Neither severe inflammation nor fibrous encapsulation was observed throughout the implantation (~12 weeks). These porous poly(l-lactic acid) membranes were highly biocompatible and functioned well as biodegradable scaffolds that could enhance the use of osteogenic periosteal sheets in therapy.

Improved adhesion of human cultured periosteal sheets to a porous poly(L‐lactic acid) membrane scaffold without the aid of exogenous adhesion biomolecules

Human cultured periosteal sheets, which are developed from small excised periosteum tissue segments (PTSs) in culture dishes by simple expansion culture, have been applied as a promising autologous osteogenic grafting material for periodontal regenerative therapy. However, the weak initial adhesion of PTSs to dish surfaces often hampers cellular outgrowth and limits the number of preparations. To correct this weakness and still avoid the use of animal-derived adhesion biomolecules, we have developed a novel, biodegradable, porous poly(L-lactic acid) (pPLLA) membrane. Freshly excised PTSs bound well to the highly porous pPLLA membrane, possibly due to the presence of semihemispheric 20-30 μm diameter openings on the upper surface. Global gene expression analysis demonstrated that periosteal sheets cultured on pPLLA membranes upregulated expression of many adhesion molecules. Osteogenic induction stimulated the production of proteoglycans by these cells and concomitantly enhanced their expansion and penetration into the deep pore regions of the membrane in parallel with the progression of in vitro mineralization. These findings suggest that our pPLLA membranes not only facilitate initial adhesion, primarily mediated by adsorbed proteins, but also enhance biological adhesion by inducing endogenous adhesion molecules in periosteal sheet cultures. Therefore, the efficacy of periosteal sheets in therapy should be greatly enhanced by using this new pPLLA membrane.

Manual cryopreservation of human alveolar periosteal tissue segments: Effects of pre-culture on recovery rate

Cultured human periosteal sheets constitute a promising grafting material for periodontal tissue regenerative therapy. However, preparation of these sheets usually requires six weeks or longer, and this lengthy commitment and delay limits both clinical applicability and availability. The aim of this study is to develop an efficient, practical, cost-effective cryopreservation method for periosteal tissue segments (PTSs). Human PTSs were aseptically excised from alveolar bone and pre-cultured in Medium 199 + 10% fetal bovine serum (FBS) for the indicated number of days before they were slowly frozen down to −75 °C in a commercial freezing vessel using medium containing 10% dimethyl sulfoxide (Me 2SO) and various concentrations of FBS. After fast-thawing at 37 °C, PTSs were again cultured, and their growth and responses to standard osteogenic induction were evaluated (vs. freshly excised PTSs). Proliferating cells were obtained at the highest levels from cryopreserved PTSs that were pre-cultured for 14 days before freezing. When a concentration of 50% or more FBS was included in the cryopreservation solution, cells migrated out more actively and grew faster. Importantly, osteoinduction up-regulated alkaline phosphatase (ALP) activity and osteoblastic marker mRNAs in cryopreserved PTS-derived sheets just as effectively as it did in native PTS-derived ones. These data suggest that pre-conditioned PTSs can be efficiently cryopreserved in a freezing solution containing high FBS by traditional manual cryopreservation methods without aid of a program freezer or more elaborate equipment.

Collagen‐Coated Poly(l‐lactide‐co‐ɛ‐caprolactone) Film: A Promising Scaffold for Cultured Periosteal Sheets

We previously demonstrated that human periosteal sheets prepared on culture dishes function as an osteogenic "graft material" applicable to periodontal regenerative therapy. However, a lower level of initial adhesion of the excised periosteum tissue segments to culture dishes was a critical point that compromised the successful preparation of functional periosteal sheets. To improve on this weakness, we developed a transparent, biodegradable poly(L-lactide-co-ɛ-caprolactone) (LCL) film and tested its function as a scaffold and carrier of periosteal sheets. Human periosteum tissue segments excised from alveolar bone of healthy donors were cultured on type I atelocollagen-coated LCL films. Initial adhesion was examined by simple agitation. Cell outgrowth and in vitro mineralization were cytohistochemically examined. Osteogenic activity was histochemically examined in an animal implantation model using nude mice. Surface collagen-coating modified the hydrophobic nature of LCL and substantially improved the initial adhesion. Compared to cultures in plastic dishes, the growth rate was delayed in non-coated films, but not in collagen-coated films. In the trimming process for animal implantation, periosteal sheets were frequently detached from non-coated films, but not from collagen-coated films. Regardless of collagen-coating, LCL films did not cause any significant infiltration of inflammatory cells, or negatively impact mineralized tissue formation. Collagen-coating improved the initial adhesion of periosteum segments, which facilitated cell outgrowth and also handling efficiency on implantation. Therefore, we believe that once evaluated in human studies, our collagen-coated LCL film will contribute to improving the periodontal regenerative methodology with the application of cultured autologous periosteal sheets.

Osteogenic activity of human periosteal sheets cultured on salmon collagen-coated ePTFE meshes

Our animal implantation studies have demonstrated that, after osteogenic processing, cultured human periosteal sheets form osteoid tissue ectopically without the aid of conventional scaffolding materials. To improve the osteogenic activity of these periosteal sheets, we have tested the effects of including a scaffold made of salmon collagen-coated ePTFE mesh. Periosteal sheets were produced with minimal manipulation without enzymatic digestion. Outgrown cells penetrated into the coated mesh fiber networks to form complex multicellular layers and increased expression of alkaline phosphatase activity in response to the osteoinduction. In vitro mineralization was notably enhanced in the original tissue segment regions, but numerous micro-mineral deposits were also formed on the coated-fiber networks. When implanted subcutaneously into nude mice, periosteal sheets efficiently form osteoid around the mineral deposits. These findings suggest that the intricate three-dimensional mesh composed of collagen-coated fibers substantially augmented the osteogenic activity of human periosteal sheets both in vitro and in vivo.

Characterization of human cultured periosteal sheets expressing bone-forming potential:in vitroandin vivoanimal studies

Our recent clinical studies have demonstrated that autologous implantation of human cultured periosteal (hCP) sheets in combination with porous hydroxylapatite (HAp) particles at the site of periodontal bone defects strikingly facilitates tissue regeneration. To better understand how the hCP sheet functions at the implantation site, we have now examined its biochemical and morphological characteristics in vitro and its ectopic osteoinductivity in nude mice. Cultured human periosteal tissue segments produced periosteal cells that migrated out from the central region within 4-8 days and grew more rapidly with longer culture times. Alkaline phosphatase activity increased in parallel with actual osteoblastic induction. Cytokine array assays demonstrated that osteoblastic induction downregulated IL-6 and thrombopoietin, but upregulated IL-8, IL-13, IGF-I and IGFBP-2 in hCP sheets. When differentiated hCP sheets were implanted alone, areas of osteoid and mineralized tissue were formed within 2 weeks, but non-induced, immature hCP sheets did not produce much mineralization. These findings suggest that mature hCP sheets potentially function not only as seeds of ectopic bone formation without the need of synthetic tissue scaffolds, but also as living drug-delivery systems, to influence cells near implantation sites by producing several important cytokines. These two major characteristics indicate that a mature hCP sheet is a promising osteoinductive biomaterial, even without conventional scaffolds for periodontal regenerative therapy.

Topographic microsurgical anatomy of the paraclinoid carotid artery.

In this publication, the authors describe the microanatomic topography of the entire paraclinoid area with respect to the paraclinoid segment of the internal carotid artery and its surrounding anatomical structures. Special attention was given to the borders of the paraclinoid area, cavernous sinus, arterial vessels, and cranial nerves passing through the region. The paraclinoid region was defined as a pyramid-formed space formed by the dural covering of the anterior clinoid process. The superior border is formed by the continuity of the anterior petroclinoid fold, anteriorly on the superior surface of the anterior clinoid process and medially in the direction of the diaphragma sellae. This dural sheet encircles the internal carotid artery and forms the so-called distal dural ring of the internal carotid artery. The medial border of the paraclinoid region is formed by the body of the sphenoid bone and the adjacent periosteal sheet. The inferior border is formed by a fibrous plate between the middle and anterior clinoid processes. This so-called proximal dural ring separates the venous compartments of the cavernous area from the paraclinoid area. The lateral border is formed by the lateral surface of the anterior clinoid process with its dural covering. The arterial supply of this region is provided by branches of the intracavernous carotid segment and the ophthalmic artery. The important nerves in close vicinity to the paraclinoidal area are the optic and the oculomotor nerves. Understanding and knowledge of the topographic anatomy of the paraclinoid area is essential for microsurgical exposure of this region.