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Delivery Systems for the BMPs   |    
Geometry of Carriers Controlling Phenotypic Expression in BMP-Induced Osteogenesis and Chondrogenesis
Yoshinori Kuboki, PhD, DDS; Qiming Jin, PhD, DDS; Hiroko Takita, PhD
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Investigation performed at Hokkaido University, Sapporo, Japan
Yoshinori Kuboki, PhD, DDS
Qiming Jin, PhD, DDS
Hiroko Takita, PhD
Department of Oral Health Science, Graduate School of Dental Science, Hokkaido University, N-13, W-7, Kita-Ku, Sapporo 060-8586, Japan

The authors did not receive grants or outside funding in support of their research or preparation of this manuscript. They did not receive payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

The Journal of Bone & Joint Surgery.  2001; 83:S105-S115 
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Abstract

Background: The effect of the geometry of extracellular matrices on bone morphogenetic protein (BMP)-induced osteogenesis has not been systematically studied. Geometry is crucially important for the scaffold in bone and joint tissue engineering. The purpose of this study was to elucidate principles of geometry of matrices in designing new scaffolds and matrices for use in reconstruction of bone and joints.

Methods: More than ten biomaterials with different geometries, including a unique device of honeycomb-shaped hydroxyapatite, were combined with BMPs of recombinant (rhBMP-2) or natural bovine origin (S300 BMP cocktail) and implanted subcutaneously into 4-week-old Wistar-King rats. The implanted pellets were removed at 1-4 weeks and analyzed for bone and cartilage formation by histological and biochemical methods.

Results: BMP-induced bone and cartilage induction was highly dependent on the geometric properties of the carrier. Some carriers such as porous particles or blocks of hydroxyapatite induced osteogenesis directly, without detectable chondrogenesis, whereas other carriers such as fibrous glass membrane induced cartilage exclusively. Still other carriers induced mostly cartilage followed by bone formation. Solid particles of hydroxyapatite and fibrous glass membrane with too tight a meshwork did not induce bone or cartilage. The optimal pore size for bone-forming efficacy in porous blocks of hydroxyapatite was a diameter of 300-400 lm. In straight tunnel structures with various diameters in honeycomb-shaped hydroxyapatite, tunnels with smaller diameters (90-120 mm) induced cartilage followed by bone formation, whereas those with larger diameters (350 mm) induced bone formation directly within the tunnels.

Conclusions: BMP carriers were classified into three types: bone-inducing, cartilage-inducing, and cartilage-bone-inducing. From the analysis of causative factors inducing osteogenesis and chondrogenesis in the BMP system, we concluded that the geometry of the carrier is crucially important and vasculature-inducing geometry should be considered in designing effective scaffolds for bone formation. We propose a classification of geometry of the artificial extracellular matrices that is useful for designing a scaffold for tissue engineering of bone and related tissues.

Clinical Relevance: Conventional requisites of the BMP carriers for clinical use have mainly concerned the affinities of carriers with cells and biomolecules and their mechanical strength. The vasculature-inducing geometry of carriers adds a new criterion in designing systems for effective bone and joint reconstruction. The geometries of porous structures—their sizes, continuity, and straightness as verified by hydroxyapatite in this study—will be applicable for other biomaterials for clinical reconstruction therapy.

Figures in this Article
    To understand the mechanisms of formation of bone and related tissues and to develop effective methods for their reconstruction, we have proposed that five factors must be taken into consideration1-5. They are (1) the cells directly involved in bone formation, (2) the matrices produced by the cells, (3) nutrients provided by vascularization, (4) regulators of general cellular activities as well as the calcification process, and (5) biomechanical dynamics. These five factors should be analyzed individually; then, the interactions between them can be elucidated and, finally, they should be integrated into the whole picture of bone formation. These principles can be applied not only to understand the mechanism of bone formation but also to reconstruct local bone defects by tissue engineering. In many clinical cases, it is not necessary to apply all five factors to the local lesion for reconstruction. Usually, the actual application of two or three factors is enough for successful reconstruction of bone and related tissues; however, care must always be taken to consider the other factors. To verify the above proposition, we chose as our experimental system bone morphogenetic protein (BMP)-induced ectopic osteogenesis6-8.
    BMP is a cytokine that induces bone formation when it is implanted with a certain carrier into ectopic tissues such as skin or muscle6,7. This ability has attracted the attention of scientists in the orthopaedic and dental fields, in anticipation of its clinical application. One of the major problems to be overcome before clinical application is the development of the optimal carriers of this cytokine3-5. Therefore, we have developed and tested more than ten different carriers (Table 1), concluding that BMP-induced osteogenesis and chondrogenesis are highly dependent on the carrier2-5,9-15. This is partly because the carrier of BMP functions not only as a drug delivery system but also as an important cell substratum on which the cells undergo growth and differentiation3,4,12-14. At the initial stage of research, BMP-induced bone formation was believed to precisely follow endochondral ossification (7). However, when new carriers of porous particles of hydroxyapatite (PPHAP) or fibrous collagen membrane (FCM) were introduced, it was found that bone was formed directly by the process of membranous ossification, without cartilage formation2-4. Furthermore, nonporous particles of hydroxyapatite did not induce bone or cartilage under the same conditions4.
    These findings led us to investigate the geometry of the BMP carrier as one of the important factors to control the efficacy of phenotypic induction in this experimental system. A series of studies revealed that there are "cartilage-inducing carriers" such as fibrous glass membrane2 and "bone-inducing carriers" such as porous particles or blocks of hydroxyapatite8 (Table 1).
    Since BMP carriers are now understood to have an important role as a cell substratum, it is necessary to reconsider the effect of matrices on cells, in vivo and in vitro, and to consider matrices of both natural and artificial origins. Conventionally, the functions of matrices have been categorized into (1) mechanical support, (2) cell substrate, (3) supply of nutrients, (4) deposition of various molecules, and (5) all other specific and general functions, including the construction of a transparent substance such as the cornea and nucleation of hydroxyapatite.
    These functions of matrices were categorized into physical, chemical, and biochemical properties, and we have now come to a new phase where we have to add a fourth: geometry. Some of the examples of functions and properties are listed in Table 2. The physical, chemical, and biochemical properties have been well studied and documented. However, the geometrical properties of extracellular matrix (ECM) have been studied by only a limited number of investigators and are poorly understood.
    In this paper, we introduce a unique BMP carrier made of honeycomb hydroxyapatite, which possesses an entirely new geometry of multiple tunnels with various diameters12, and discuss the results together with recently published studies on the geometry of artificial ECM.
     
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    +Fig. 1-A:Figure 1 Apparatus for the preparation of honeycomb hydroxyapatite (A) and its products (B and C). A paste of hydroxyapatite powder was pushed out through the multi-nozzle disc, in the form of multiple rods, into a condensation space that was constructed in the form of taped conical tubing. From the outer surface of the nozzle disc, multiple guiding sticks were extruded perpendicularly toward the conical tubing, which created longitudinal holes in the hydroxyapatite paste. The paste was gradually reduced in diameter as it was pushed through the conical tubing (A). The honeycomb-shaped cylindrical products (B and C) were sintered at 1,000°C and cut to the required lengths. Honeycomb apatite implants with a small tunnel diameter (120 mm, seven tunnels) (B) and a large tunnel diameter (350 m, 19 tunnels) (C) are shown. Bars in B and C indicate 200 m.
     
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    +Fig. 1-B:Figure 1 Apparatus for the preparation of honeycomb hydroxyapatite (A) and its products (B and C). A paste of hydroxyapatite powder was pushed out through the multi-nozzle disc, in the form of multiple rods, into a condensation space that was constructed in the form of taped conical tubing. From the outer surface of the nozzle disc, multiple guiding sticks were extruded perpendicularly toward the conical tubing, which created longitudinal holes in the hydroxyapatite paste. The paste was gradually reduced in diameter as it was pushed through the conical tubing (A). The honeycomb-shaped cylindrical products (B and C) were sintered at 1,000°C and cut to the required lengths. Honeycomb apatite implants with a small tunnel diameter (120 mm, seven tunnels) (B) and a large tunnel diameter (350 m, 19 tunnels) (C) are shown. Bars in B and C indicate 200 m.
     
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    +Fig. 1-C:Figure 1 Apparatus for the preparation of honeycomb hydroxyapatite (A) and its products (B and C). A paste of hydroxyapatite powder was pushed out through the multi-nozzle disc, in the form of multiple rods, into a condensation space that was constructed in the form of taped conical tubing. From the outer surface of the nozzle disc, multiple guiding sticks were extruded perpendicularly toward the conical tubing, which created longitudinal holes in the hydroxyapatite paste. The paste was gradually reduced in diameter as it was pushed through the conical tubing (A). The honeycomb-shaped cylindrical products (B and C) were sintered at 1,000°C and cut to the required lengths. Honeycomb apatite implants with a small tunnel diameter (120 mm, seven tunnels) (B) and a large tunnel diameter (350 m, 19 tunnels) (C) are shown. Bars in B and C indicate 200 m.
     
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    +Fig. 2:Figure 2 Geometry-directed cartilage and bone formation in honeycomb hydroxyapatite (HCHAP) combined with recombinant human bone morphogenetic protein-2 (rhBMP-2) in the smaller diameter tunnel (90-120 m). In these tunnels, at 1 week after implantation young mesenchymal cells migrated into the tunnels of HCHAP and some cartilage appeared between the HCHAP (A). At 2-3 weeks, active chondrogenesis filled the space within the tunnels (B). Vascularization and osteogenesis started to develop in the tunnels. At 3 weeks, osteogenesis began from the orifice and developed toward central areas, forming bone on the inner surfaces of the tunnels (C). At 4 weeks, cartilage disappeared and was replaced by bone (D). Bars indicate 300 m.
     
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    +Fig. 3:Figure 3 Geometry-directed cartilage and bone formation in honeycomb hydroxyapatite (HCHAP) combined with recombinant human bone morphogenetic protein (rhBMP-2) in the larger diameter tunnel (350 m). In these tunnels, at 1 week the condensation of mesenchymal cells started accompanied by vasculature (A), but no cartilage was observed throughout the experimental periods. Instead, osteoid-like tissues and bone tissues appeared dispersed within the tunnels and developed into the larger mass of bone at 2-3 weeks (B). At 4 weeks, thick layers of bone were observed along the inner surface of the tunnels. In the central area of the tunnels, bone-marrow-like tissue that contained numerous adipocytes and young blood cells was clearly observed (C and D). Importantly, in both types of HCHAP, capillaries developed throughout the tunnels, eventually creating a structure similar to the Haversian system of bone remodeling. Bars indicate 100 m.
     
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    +Fig. 4-A:Figure 4 Schematic representation of geometry-directed chondrogenesis and osteogenesis in the honeycomb hydroxyapatite with larger (A) and smaller (B) tunnels, which are applied as bone morphogenetic protein (BMP) carriers. In the smaller tunnels (90-110 m in diameter), cartilage was formed followed by bone formation, similar to the process of endochondral ossification. In contrast, in the larger tunnels (350 m in diameter) bone was formed directly without detectable cartilage formation. Figure 4-A reprinted, with permission, from: Kuboki Y, Takita H, Mizuno M, Fujisawa F: Geometry of artificial extracellular matrices: a new paradigm from dental tissue engineering. Dentistry in Japan. 2001;37:101.
     
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    +Fig. 4-B:Figure 4 Schematic representation of geometry-directed chondrogenesis and osteogenesis in the honeycomb hydroxyapatite with larger (A) and smaller (B) tunnels, which are applied as bone morphogenetic protein (BMP) carriers. In the smaller tunnels (90-110 m in diameter), cartilage was formed followed by bone formation, similar to the process of endochondral ossification. In contrast, in the larger tunnels (350 m in diameter) bone was formed directly without detectable cartilage formation. Figure 4-A reprinted, with permission, from: Kuboki Y, Takita H, Mizuno M, Fujisawa F: Geometry of artificial extracellular matrices: a new paradigm from dental tissue engineering. Dentistry in Japan. 2001;37:101.
     
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    +Fig. 5-A:Figure 5 Alkaline phosphatase activity (A) and osteocalcin contents (B) of implants of bone morphogenetic protein combined with porous blocks of hydroxyapatite of different pore sizes.
     
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    +Fig. 5-B:Figure 5 Alkaline phosphatase activity (A) and osteocalcin contents (B) of implants of bone morphogenetic protein combined with porous blocks of hydroxyapatite of different pore sizes.
     
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    +Fig. 6:Figure 6 Microphotographs of cross sections of bone morphogenetic protein (BMP)-combined porous blocks of hydroxyapatite 4 weeks after implantation. Pore sizes are 106-212 m in (A) and (B), 212-300 m in (C) and (D), 300-400 m in (E) and (F), 400-500 m in (G) and (H), and 500-600 m in (I) and (J). Original magnifications of photographs are 75 in the right lane and 150 in the left lane. Bars in the lower right corners of (I) and (J) indicate 100 m. Pores are shown as multiple round structures in all photographs (indicated by PO in A, for example). The spaces other than the pores (indicated by HAP in D, for example) have been occupied by hydroxyapatite. Bone formation was observed from the inner surface of almost all the pores (indicated by BO in F, for example) smaller than 500 m. On the other hand, bone not only formed along the inner surface of the pore but occupied the inner space (indicated by an arrow in I) in 500-600-m pores. Reprinted, with permission, from: Tsuruga E, Takita H, Itoh H, Wakisaka Y, Kuboki Y. Pore size of porous hydroxyapatite as the cell-substratum controls BMP-induced osteogenesis. J Biochem. 1997:121:317-24.
     
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    +Fig. 7:Classification of geometry in extracellular matrices. Reprinted, with permission, from: Kuboki Y, Takita H, Mizuno M, Fujisawa F: Geometry of artificial extracellular matrices: a new paradigm from dental tissue engineering. Dentistry in Japan. 2001;37:101.
     
    Anchor for JumpAnchor for Jump  TABLE 1 Taxonomy of bone morphogenetic protein (BMP) carriers
    CarrierAbbreviationReference(s)
    Bone-inducing
    Porous particles of hydroxyapatitePPHAP2,4
    Porous blocks of hydroxyapatitePBHAP3
    Laser-perforated collagen membraneLPM14
    Cartilage-inducing
    Fibrous glass membraneFGM2,10
    Bone and cartilage-inducing
    Insoluble bone matrixIBM12
    Fibrous collagen membraneFCM5,11
    Collagen beadsCB16,17
    Fibrous mass of new bio-glassCPSA13
    Titanium meshTM18
    Honeycomb hydroxyapatiteHCHAP12
     
    Anchor for JumpAnchor for Jump  TABLE 2 Functions and properties of extracellular matrices
    PropertiesFunctions
    PhysicalMechanical support for cells, tissues, organs, and body
    ChemicalSupport of cells and molecules through surface charges; supply and deposition of various molecules
    BiochemicalSpecific interactions among adhesion molecules, signaling molecules (cytokines), and cell surface receptors
    GeometricalDirecting growth and differentiation of cells and tissues

    Honeycomb-Shaped Hydroxyapatite (HCHAP)

    HCHAP was prepared with a special nozzle device (Figs 1-A, 1-B, and 1-C)12. A paste of hydroxyapatite powder was extruded through a multi-nozzled disc, in the form of multiple rods, into a condensation space of taped conical tubing. From the outer surface of the nozzled disc, multiple guiding sticks were extruded perpendicularly toward the conical tubing, which created longitudinal holes in the transporting hydroxyapatite paste. The paste was gradually reduced in diameter as it was pushed through the conical tubing (Fig. 1-A). The cylindrical products were sintered at 1,000°C and cut to the required lengths. The sizes of the cylindrical products, including tunnel diameters, were controlled. Two products with large or small tunnels were used in this study. Cylindrical particles of HCHAP (0.7 1.0-1.5 mm) with smaller tunnel diameters (90-120 m, seven tunnels) and particles (3 1.0-1.5 mm) with a larger tunnel diameter (350 m, 19 tunnels) were compared. The tunnels ran through the particles longitudinally. The scanning electron microscopic (SEM) patterns of HCHAP are shown in Figs. 1-B and 1-C.

    Implantations

    A solution (5 mg/30 ml) of recombinant human BMP-2 (rhBMP-2) (a kind gift from Yamanouchi Co., Japan) was absorbed into the two sorts of HCHAP carriers (40 mg) with small or large tunnels. The carrier/rhBMP-2 composites were immediately lyophilized. Four-week-old rats (Wistar, male) were anesthetized with pentobarbital sodium (4 mg/100 g body weight), and the samples were implanted subcutaneously into the backs of rats. Different rats were used to examine implantation of four samples of the same carrier/rhBMP-2 composites. After weeks 1, 2, 3, and 4, the samples were removed for examination2-5,9-14.

    Histological Observations

    For histological observation, the implanted samples were carefully removed from the tissue as a single pellet, fixed in 10% neutral formaldehyde, and decalcified in Blank-Rychlo solution at 4°C for 5 hours. After being washed with water, the pellets were dehydrated with gradient alcohol and embedded in paraffin. Finally, the samples were cut into 4-5 mm sections. The sections were stained with hematoxylin and eosin routinely, as well as with toluidine blue.

    Honeycomb Hydroxyapatite

    Figure 2 shows histological profiles of the BMP-containing honeycomb-shaped hydroxyapatite (HCHAP) with smaller tunnels (90-120 mm in diameter), removed from the tissues at 1 week. Mesenchymal cells penetrated into the whole implant and condensed on the surface of HCHAP and the orifice area of the tunnels. Chondrogenesis had already occurred within the tunnels and between HCHAP particles. No bone formation was observed at this stage (Fig. 2-A). In contrast, at 2 weeks, bone formation was clearly seen over the outer surface of HCHAP and orifice areas of the tunnels (Fig. 2-B). Cartilage was seen within the tunnels (Fig. 2-C). Bone was typically seen near the orifice areas within the tunnels, whereas cartilage was located in the middle portion of the tunnel continuously to bone areas. Thus, the cartilage at both ends of the tunnel appeared to be replaced by bone, whereas there was still cartilage in the center of the tunnel at 2-3 weeks. At 4 weeks, the cartilage was totally replaced by bone and had disappeared. Bone formed not only on the surfaces of tunnels but also between HCHAP particles (Fig. 2-D). Vascularization could be seen clearly in the tunnels. A structure similar to a Haversian system was observed in some tunnels of HCHAP particles. These processes were very similar to those previously reported12.
    The HCHAP with 350-mm tunnels induced quite a different process of osteogenesis. At 1 week, the condensation of mesenchymal cells was accompanied by vasculature (Fig. 3-A), but no cartilage appeared throughout the experimental periods. Instead, osteoid-like tissues and bone tissues appeared dispersed within the tunnels and developed into a larger mass of bone at 2 weeks (Fig. 3-B). At 4 weeks, thick layers of bone were observed along the inner surface of the tunnels. In the central area of the tunnels, bone marrow that contained numerous adipocytes and young blood cells was clearly observed (Figs. 3-C and 3-D). These results are schematically described in Figs. 4-A and 4-B.

    Porous Blocks of Hydroxyapatite3 (Reproduced with the Permission of the Japanese Biochemical Society)

    When we studied the BMP carrier of the porous block of hydroxyapatite (PBHAP) with a series of different pore sizes, the highest efficacy of bone formation was observed in the PBHAP with pores of 300-400 mm as judged from alkaline phosphatase (ALP) activities and osteocalcin contents (Figs. 5-A and 5-B)3. ALP activities of implants with 300-400-mm pores were 3.5 times higher than those of implants with 106-212-mm pores (Fig. 5-A). Osteocalcin contents of implants with 300-400-mm pores were 2.0 times higher than those of implants with 106-212-mm pores. Osteocalcin contents of the implants with pore sizes larger than 300 mm were significantly higher than those of implants with 106-212-mm pores (Fig. 5-B).
    Histologically, the implants with different-sized pores showed similar changes until 2 weeks after implantation. The pores were commonly filled with loose fibrous connective tissue at 1 week, and the fibrous structure became more compact at 2 weeks. At 3 weeks, bone formation was histologically observed on the inner surface of the pores. At 4 weeks, we observed the histological characteristics of bone in the ceramics of all pore sizes. In the ceramics with 106-212-mm pores (Figs. 6-A and 6-B), we observed the bone structure on the surface of pores accompanied by cement lines, indicating that bone remodeling occurred in this stage. Bone did not form in some pores. In the ceramics with 212-300-mm pores (Figs. 6-C and 6-D), almost all pores were filled with bone. Interestingly, there was always a certain space for vascular tissue within a pore. This characteristic was quite similar to what we observed in the porous particles of hydroxyapatite with an average pore size of 150 mm2. In the ceramics with 300-400 mm (Figs. 6-E and 6-F) and 400-500 mm pores (Figs. 6-G and 6-H), bone marrow cells and adipocytes within the pores accompanied bone tissues. In some pores, we could distinguish both capillaries and bone marrow cells side by side within the same space surrounded by bone. In the ceramics with 500-600-mm pores, bone formed not only along the inner surface of the pore but also occupied the inner space of the pore. Finally, a certain mechanical stability seemed to be required for maintaining the inner wall of the pore that is constructed by bone. As the pore became larger than 300-400 mm, the inner wall became thinner (Fig. 3). In the ceramics with a pore-size range of 500-600 mm, there was no more integrated formation of bone along the inner walls (Figs. 6-I and 6-J). Bone formation in the 500-600-mm pores occupied the area inside the pores, and the rigid lining along the inner surface was interrupted (Figs. 6-I and 6-J). In the 400-500 and 500-600-mm pores, multiple and larger capillaries appeared within a single pore; this was not observed in the pores smaller than 300-400 mm. These observations suggested that a pore size of 300-400 mm was most appropriate for Haversian-type bone formation, leading to preferential formation of bone compared with the other pore sizes3.

    Historical Background of Studies on the Geometry of Matrices

    Reddi and Huggins3 first showed the effects of matrices on cell differentiation. They demonstrated that the matrices of open-tube and dead-end tube structures had different effects on osteogenesis and chondrogenesis inside the tubes19. These two geometrically different open and dead-end tubes were prepared by use of the middle and apical portions of rat incisor dentin, respectively. They were packed with decalcified bone particles (which contained BMP) and were implanted in rat skin. The results showed that the open tube induced osteogenesis and the dead-end tube induced chondrogenesis. Reddi and Huggins interpreted the results to be due to the higher vasculature in the open tube, so that the higher supply of oxygen and nutrients favored osteogenesis whereas the lesser vasculature in the dead-end tube led to chondrogenesis. Their report was also one of the first to describe the effect of the microenvironment created by the ECM19.
    Later, Sampath and Reddi20 reported that a coarse powder (420-850-mm particle size) of demineralized bone induced a much higher yield of bone than a fine powder (44-74 mm) when it was used as a carrier for BMP-induced bone formation. Van Eeden and Ripamonti21 reported the effects of the geometry of the carrier on bone formation by comparing porous hydroxyapatite in block and granular forms with different pore sizes (200 and 500 mm). They reported much higher efficacy of bone induction in the block than in the granular form. However, these authors did not mention the vasculature as a causative factor of differentiation into chondrogenesis and osteogenesis.

    Detection of Direct Bone Formation in the Vasculature-Inducing Geometry

    On the other hand, direct bone formation without chondrogenesis occurred in the carrier of porous particles of hydroxyapatite (PPHAP) combined with BMP2,4. This direct bone formation was explained by rapid vascularization through the interconnected pores in the PPHAP, which did not provide the hypoxic microenvironment necessary for chondrogenesis. Recently, Murata et al.15 demonstrated the carrier dependency of cellular differentiation using PPHAP combined with BMP and confirmed that the PPHAP/BMP composite induced only direct bone formation without chondrogenesis. Sasano et al.9 also showed direct bone formation in the local area within the carrier, independent of endochondral ossification, using a fibrous collagen membrane (FCM) with BMP.
    Kuboki et al.4 compared the efficacies of three hydroxyapatite ceramics with different geometrical structures as carriers for BMP-induced osteogenesis. In their experiments, solid nonporous particles, coral-replicated porous disc of hydroxyapatite (5 mm in diameter and 2 mm in height; Interpore International, Irvine, California) and PPHAP, were subcutaneously implanted into rats. The results indicated that PPHAP and coral-HAP induced osteogenesis effectively due to the geometry of the interconnected porous structures that created spaces for vasculature. On the other hand, solid nonporous particles did not induce osteogenesis or chondrogenesis because the smooth surface structure and close contacts of the particles inhibited vascular development and proliferation of mesenchymal cells. They proposed that a feasible geometry of hydroxyapatite for osteogenesis is interconnected porosity that leads to vascularization.

    Geometry Created by Fibrous Glass Membrane: Vasculature-Inhibiting Geometry

    Kuboki et al.2 demonstrated the effects of geometrical factors of matrices on cell differentiation using two distinctive BMP carriers: fibrous glass membrane (FGM) and PPHAP, which induced zonal chondrogenesis and membranous ossification, respectively. The FGM, an unwoven sheet of glass fibers (fibril diameter, 1 mm; thickness, 1 mm), in combination with BMP exclusively produced cartilage within the membrane when implanted in rat skin. This result was explained by the finding that FGM prevented capillary invasion while it allowed young mesenchymal cells to enter, proliferate, and differentiate into chondrocytes. However, over a prolonged time, the network structure of FGM was gradually degraded and accepted vascular development and finally cartilage was replaced by bone10. When the same glass fibers were molded into a ball, combined with BMP, and implanted in rat skin, they did not induce cartilage or bone (data not shown). This was probably due to the small pore sizes within the ball, which excluded cellular migration and vasculature as well. Recently, Takita et al.22showed that the same FGM combined with recombinant human basic fibroblast growth factor (rh-bFGF) and rhBMP-2 enhanced osteogenesis at 2 weeks while decreasing cartilage formation, probably due to the increasing effect of rh-bFGF on vascularization.

    Verification of Vasculature in Porous and Nonporous Carriers of CPSA Glass Fibers

    From the histological observations of the bone tissues in the pores of hydroxyapatite, it was demonstrated that a certain small space in the central area of each pore always remained for blood vessels. This indicated that the BMP-induced bone formation was always accompanied by the blood vessels, leading to the confirmation that vasculature and bone formation are highly related2,3,22-25. A recent work by Mahmood et al.13 clearly showed that the receptors of vascular endothelial growth factor, Flt-1 and KDR23, appeared to be accompanied by bone formation in a BMP carrier of the bio-glass fibers called CPSA (CaO, P2O5, SiO2, and Al2O3). These CPSA glass fibers, which have a thicker diameter (9 mm) than that of FGM (1 mm), could be molded into a ball-shaped porous BMP carrier. This induced the typical cartilage-bone-type osteogenesis in which Flt-1 and KDR clearly appeared. In contrast, when the glass fibers were bundled to form a nonporous structure, the carrier did not induce osteogenesis significantly and Flt-1 and KDR were not detected. Thus, it was suggested that these receptors of vascular endothelial growth factor could be new candidates for the biochemical marker of local osteogenesis.

    What Is the Optimal Size of Pores?

    Various shapes of hydroxyapatite have been proved to be compatible cell substrata for osteogenic cells2,3,26,27. For local orthotopic bone reconstruction, implantation of a porous form of hydroxyapatite has generally been considered to be more feasible than nonporous particles or solids; however, there is no systematic and definitive evidence concerning the (ideal) appropriate geometry of carrier for bone formation. A study by Tsuruga et al.3 demonstrated for the first time that, under certain circumstances, a pore size of 300-400 mm was most effective for bone formation (Figs. 5 and 6). Interestingly, the morphology of bone formation in the pores of PBHAP and also of the PPHAP resembled that of the Haversian system in bone remodeling. The average diameter in the Haversian system (approximately 300 mm) coincided with the optimal size shown in this study, indicating the existence of an essential and optimal unit of bone formation of this size. These results led us to a concept of optimal curvature of the matrix surfaces that is ideal for osteoblast growth and differentiation.

    Vasculature Induction by the Biomimetic Tunnels, HCHAP

    More recently, a new BMP carrier, honeycomb-shaped hydroxyapatite (HCHAP), confirmed the idea of tubing geometry for vascularization12. This BMP carrier, with multiple tunnels, numbers, and sizes that are controllable, resulted in biomimetic processes of endochondral ossification and also of Haversian-type bone remodeling (Figs. 1 and 2). A previous study had dealt only with HCHAP with a tunnel diameter of 110 mm12. In the present experiment, the osteogenic processes of small and large tunnels, 90-120 and 350 mm, were compared.
    Unlike other hydroxyapatite BMP carriers, cartilage first filled the space within the tunnel at 1-2 weeks. The cartilage was then replaced by vascular invasion along with the bone formation on the inner surfaces of the tunnels (Fig. 2). This was considered to be due to a delay of vascular development throughout the longer length of the tunnel12. However, with the larger tunnel diameter of 350 mm, there might be enough spaces for vascularization from the onset, leading to the direct bone formation without cartilage formation detected (Fig. 3). These results further confirmed the previous conclusion that the geometry of the BMP carrier controls the differentiation into chondrogenesis and osteogenesis.
    The carrier-dependent osteogenesis and chondrogenesis in the previously described reports may be attributed to high oxygen tension and nutrients provided by the increased vascularization in the feasible geometry, which favor osteogenesis, whereas low oxygen tension favors chondrogenesis, a fact verified historically by Bassett and Herrmann28. Thus, we propose a general rule: BMP, if combined with a carrier in which vascular invasion is geometrically feasible, can induce immature cells to become bone-forming cells that cause direct bone formation. On the other hand, BMP combined with a carrier for which vascular invasion is geometrically less feasible first induces cartilage, which eventually leads to bone formation as capillaries grow. This explanation was partly verified by the fact that mRNA expression of the receptors of vascular endothelial growth factor, KDR and Flt-123-25, was extremely high in the bone-inducing BMP carriers with porous structures but not in nonporous ones13. Thus, it was concluded that the porous structure of adequate geometry is closely related to the growth rate of vascularization and thereby with bone formation.

    Concave Geometries Feasible for Cell Growth and Bone Formation

    Another interesting aspect of the geometric factor of matrices is the concave structure on the flat surface. Let us examine two examples. Photolithographic technology enables the creation of microgrooves or pits on the micrometer order on the surface of silicon monocrystal plates. With this technology, we have created numerous micropits, from 25 to 600 mm, on silicon monocrystal plates29. The surfaces of the plates were treated with collagen solution for use in cell culture. Cultures of osteoblastic cells from rat bone marrow provided meaningful results. The optimal size for cell growth was found in square micropits of 50 mm29. Ripamonti et al.30 reported a series of experiments in which porous hydroxyapatite showed bone-inductive activity when it was implanted ectopically into baboon muscles. They pointed out the possible accumulation of endogenous BMPs on the surface of hydroxyapatite in explaining the mechanism of this interesting phenomenon. Furthermore, they reported that osteogenesis was observed only in the concave surfaces of the implants, leading them to another investigation. They created concavities with the diameters of 800-1,600 mm on the surfaces of monolithic discs of hydroxyapatite; this process induced local osteogenesis as expected30.
    The concavity created by Ripamonti et al., the micropits studied by our group, and the microgrooved surface devised by den Braber et al.31 may have a common functional mechanism. The mechanisms behind the osteogenesis in the concavity and the effects of micropits on cell growth need an explanation other than vasculature (for example, the reactions such as the local concentration of cytokines or the activation of the cytoskeletons). In these structures, the size of concavities (curvature) seems to be important because a concavity that is too large may become close to the flat plane and one that is too small may limit the cell activities. Our tentative conclusion is that micropits with 50-m diameters show the maximum growth effect on bone marrow stromal cells. Confirming this will require additional experiments with cells of other origins.

    Conclusions: Taxonomy of Geometric Properties

    We have emphasized the importance of the geometry of artificial ECM for tissue engineering. Now it is appropriate to classify these specific geometries. The table in Fig. 7 summarizes the taxonomy of geometries in the artificial ECM. Most of them were verified by our group and in the literature. We have attempted to classify extracellular geometry into three basic categories: convex, planar, and concave. In each category, there are several fundamental characteristic shapes. Concave geometry, for instance, has pores, tunnels, concavities, micropits, and grooves. Individual examples of BMP carriers or cell substrata of each fundamental shape are shown with references.
    One useful feature of this table is that we can anticipate to a certain degree what results may be obtained as a BMP carrier or cell substratum when a new material is introduced. At the very least, comparative discussions concerning the different scaffolds may become easier with this kind of table. For instance, pores and tunnels and concavities show remarkable similarities in bone-forming ability. In future tissue engineering, numerous variations and mixed-type shapes of these three categories and ten fundamental shapes must be developed. With discussions and insights from this kind of whole picture, we can predict and even design efficacies of new scaffolds.
    Kuboki Y, Yamaguchi H, Yokoyama A, Murata M, Takita H, Tazaki M, Mizuno M, Hasegawa T, Iida S, Shigenobu K, Fujisawa R, Kawamura M, Atsuta T, Matsumoto A, Kato H, Zhou H-Y, Ono I, Takeshita N, Nagai N. The bone-biomaterials interface. Osteogenesis induced by BMP-coated biomaterials. Biochemical principles of bone reconstruction in dentistry. In: The Bone-Biomaterial Interface, University of Toronto Press, Toronto, p 127-38, 1991 
     
    KubokiY, Saito T, Murata M, Takita H, Mizuno M, Inoue M, Nagai N,Poole AR. Two distinctive BMP-carriers induce zonal chondrogenesis and membranous ossification, respectively; geometrical factors of matrices for cell-differentiation. Connect Tissue Res,1995;32: 219-26. 32219  1995  [PubMed]
     
    TsurugaE, Takita H, Itoh H, Wakisaka Y,Kuboki Y. Pore size of porous hydroxyapatite as the cell-substratum controls BMP-induced osteogenesis. . J Biochem,1997;121: 317-24. 121317  1997  [PubMed]
     
    KubokiY, Takita H, Kobayashi D, Tsuruga E, Inoue M, Murata M, Nagai N, Dohi Y,Ohgushi H. BMP-induced osteogenesis on the surface of hydroxyapatite with geometrically feasible and nonfeasible structures: topology of osteogenesis. J Biomed Mater Res,1998;39: 190-9. 39190  1998  [PubMed]
     
    KubokiY, Sasaki M, Saito A, Takita H,Kato H. Regeneration of periodontal ligament and cementum by BMP-applied tissue engineering. Eur J Oral Sci,1998;106 Suppl 1: 197-203. 106 Suppl 1197  1998  [PubMed]
     
    UristMR. Bone: formation by autoinduction. Science,1965;150: 893-9. 150893  1965  [PubMed]
     
    ReddiAH. Cell biology and biochemistry of endochondral bone development. Coll Relat Res,1981;1: 209-26. 1209  1981  [PubMed]
     
    WozneyJM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, Hewick RM,Wang EA. Novel regulators of bone formation: molecular clones and activities. Science,242: 1528-34. 1988;2421528  1988  [PubMed]
     
    SasanoY, Ohtani E, Narita K, Kagayama M, Murata M, Saito T, Shigenobu K, Takita H,, Mizuno M,Kuboki Y. BMPs induce direct bone formation in ectopic sites independent of the endochondral ossification in vivo. Anat Rec,1993;236: 373-80. 236373  1993  [PubMed]
     
    SasanoY, Mizoguchi I, Takahashi I, Kagayama M, Saito T,Kuboki Y. BMPs induce endochondral ossification in rats when implanted ectopically within a carrier made of fibrous glass membrane. Anat Rec,1997;247: 472-8. 247472  1997  [PubMed]
     
    KobayashiD, Takita H, Mizuno M, Totsuka Y,Kuboki Y. Time-dependent expression of bone sialoprotein fragments in osteogenesis induced by bone morphogenetic protein. J Biochem. ,1996;119: 475-81. 119475  1996  [PubMed]
     
    JinQM, Takita H, Kohgo T, Atsumi K, Itoh H,Kuboki Y. Effects of geometry of hydroxyapatite as a cell substratum in BMP-induced ectopic bone formation. J Biomed Mater Res,2000;51: 491-9. 51491  2000  [PubMed]
     
    MahmoodJ, Takita H, Ojima Y, Kobayashi M, Kohgo T,Kuboki Y. Geometric effect of matrix upon cell differentiation: BMP-induced osteogenesis using a new bioglass with a feasible structure. J Biochem,2001;129: 163-71. 129163  2001  [PubMed]
     
    KikuchiM, Takita H, Nakayama Y, Matsuda T,Kuboki Y. Laser-perforated collagen membrane: pore size-dependent bone induction of a new BMP carrier. J Hard Tissue Biol,9: 2000;in press9  2000 
     
    MurataM, Inoue M, Arisue M, Kuboki Y,Nagai N. Carrier-dependency of cellular differentiation induced by bone morphogenetic protein in ectopic sites. Int J Oral Maxillofac Surg,1998;27: 391-6. 27391  1998  [PubMed]
     
    YokoyamaA. Experimental study of bone morphogenetic protein-purification and application for bony defect. Hokkaido J Oral Biology,1990;10: 151-84. 10151  1990 
     
    MizunoM, Kasagi T,Kuboki Y. Cross-linked collagen gel shares as a useful carrier for cell culture of MC3T3-E1 clonal osteogenic cells. Jpn J Oral Biol,1988;30: 855-853. 30855  1988 
     
    KubokiY, Takita H, Tsuruga E, Ono M,Jansen JA. Rationale for hydroxyapatite-coated titanium-mesh as an effective carrier for BMP. Dent Res,1988;77: 263. 77263  1988 
     
    ReddiAH,Huggins CB. Influence of geometry of transplanted tooth and bone on transformation of fibroblast. Proc Soc Exp Biol Med. ,1973;143: 634-7. 143634  1973  [PubMed]
     
    SampathTK,Reddi AH. Importance of geometry of the extracellular matrix in endochondral bone differentiation. J Cell Biol,1984;98: 2192-7. 982192  1984  [PubMed]
     
    van EedenSP,Ripamonti U. Bone differentiation in porous hydroxyapatite in baboons is regulated by the geometry of the substratum: implications for reconstructive craniofacial surgery. Plast Reconstr Surg,1994;959-66. 959  1994 
     
    TakitaH, Tsuruga E, Ono I,Kuboki Y. Enhancement by bFGF of osteogenesis induced by rhBMP-2 in rats. Eur J Oral Sci,1997;105: 588-92. 105588  1997  [PubMed]
     
    Yeh L-C,Lee JC. Osteogenic protein-1 increases gene expression of vascular endothelial growth factor in primary cultures of fetal rat calvaria cells. Mol Cell Endocrinol,1999;153: 113-24. 153113  1999  [PubMed]
     
    ParfittAM. The mechanism of coupling: a role for the vasculature. Bone,2000;26: 319-32. 26319  2000  [PubMed]
     
    TruetaJ. The role of the vessels in osteogenesis. J Bone Joint Surg Br,1993;45: 402-18. 45402  1993 
     
    OnoI, Ohura T, Murata M, Yamaguchi H, Ohnuma Y,Kuboki Y. A study on bone induction in hydroxyapatite combined with bone morphogenetic protein. Plast Reconst Surg,1992;90: 870-9. 90870  1992  [PubMed]
     
    OnoI, Gunji H, Kaneko F, Saito T,Kuboki Y. Efficacy of hydroxyapatite ceramic as a carrier for recombinant human bone morphogenetic protein. . J Craniofac Surg,1995;6: 238-44. 6238  1995  [PubMed]
     
    BassettAC,Herrmann I. Influence of oxygen concentration and mechanical factors on differentiation of connective tissues in vitro. Nature,1961;190: 4660-1. 1904660  1961 
     
    Kikuchi H, Kikuchi Y, Kuboki Y. Micropit effect on the growth of osteoblast-like cells studied using microfabricated pit arrays. Presented at the third annual meeting of the Japanese Tissue Engineering Society, Hiroshima, 2000 
     
    RipamontiU, Crooks J,Kirkbride AN. Sintered porous hydroxyapatites with intrinsic osteoinductive activity: geometric induction of bone formation. S Afr Sci,1999;95: 335-43. 95335  1999 
     
    den BraberET, de Ruijter JE, Ginsel LA, von Recum AF,Jansen JA. Orientation of ECM protein deposition, fibroblast cytoskeleton, and attachment complex components on silicone microgrooved surfaces. J Biomed Mater Res,1998;40: 291-300. 40291  1998  [PubMed]
     

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    Anchor for JumpAnchor for Jump
    +Fig. 1-A:Figure 1 Apparatus for the preparation of honeycomb hydroxyapatite (A) and its products (B and C). A paste of hydroxyapatite powder was pushed out through the multi-nozzle disc, in the form of multiple rods, into a condensation space that was constructed in the form of taped conical tubing. From the outer surface of the nozzle disc, multiple guiding sticks were extruded perpendicularly toward the conical tubing, which created longitudinal holes in the hydroxyapatite paste. The paste was gradually reduced in diameter as it was pushed through the conical tubing (A). The honeycomb-shaped cylindrical products (B and C) were sintered at 1,000°C and cut to the required lengths. Honeycomb apatite implants with a small tunnel diameter (120 mm, seven tunnels) (B) and a large tunnel diameter (350 m, 19 tunnels) (C) are shown. Bars in B and C indicate 200 m.
    Anchor for JumpAnchor for Jump
    +Fig. 1-B:Figure 1 Apparatus for the preparation of honeycomb hydroxyapatite (A) and its products (B and C). A paste of hydroxyapatite powder was pushed out through the multi-nozzle disc, in the form of multiple rods, into a condensation space that was constructed in the form of taped conical tubing. From the outer surface of the nozzle disc, multiple guiding sticks were extruded perpendicularly toward the conical tubing, which created longitudinal holes in the hydroxyapatite paste. The paste was gradually reduced in diameter as it was pushed through the conical tubing (A). The honeycomb-shaped cylindrical products (B and C) were sintered at 1,000°C and cut to the required lengths. Honeycomb apatite implants with a small tunnel diameter (120 mm, seven tunnels) (B) and a large tunnel diameter (350 m, 19 tunnels) (C) are shown. Bars in B and C indicate 200 m.
    Anchor for JumpAnchor for Jump
    +Fig. 1-C:Figure 1 Apparatus for the preparation of honeycomb hydroxyapatite (A) and its products (B and C). A paste of hydroxyapatite powder was pushed out through the multi-nozzle disc, in the form of multiple rods, into a condensation space that was constructed in the form of taped conical tubing. From the outer surface of the nozzle disc, multiple guiding sticks were extruded perpendicularly toward the conical tubing, which created longitudinal holes in the hydroxyapatite paste. The paste was gradually reduced in diameter as it was pushed through the conical tubing (A). The honeycomb-shaped cylindrical products (B and C) were sintered at 1,000°C and cut to the required lengths. Honeycomb apatite implants with a small tunnel diameter (120 mm, seven tunnels) (B) and a large tunnel diameter (350 m, 19 tunnels) (C) are shown. Bars in B and C indicate 200 m.
    Anchor for JumpAnchor for Jump
    +Fig. 2:Figure 2 Geometry-directed cartilage and bone formation in honeycomb hydroxyapatite (HCHAP) combined with recombinant human bone morphogenetic protein-2 (rhBMP-2) in the smaller diameter tunnel (90-120 m). In these tunnels, at 1 week after implantation young mesenchymal cells migrated into the tunnels of HCHAP and some cartilage appeared between the HCHAP (A). At 2-3 weeks, active chondrogenesis filled the space within the tunnels (B). Vascularization and osteogenesis started to develop in the tunnels. At 3 weeks, osteogenesis began from the orifice and developed toward central areas, forming bone on the inner surfaces of the tunnels (C). At 4 weeks, cartilage disappeared and was replaced by bone (D). Bars indicate 300 m.
    Anchor for JumpAnchor for Jump
    +Fig. 3:Figure 3 Geometry-directed cartilage and bone formation in honeycomb hydroxyapatite (HCHAP) combined with recombinant human bone morphogenetic protein (rhBMP-2) in the larger diameter tunnel (350 m). In these tunnels, at 1 week the condensation of mesenchymal cells started accompanied by vasculature (A), but no cartilage was observed throughout the experimental periods. Instead, osteoid-like tissues and bone tissues appeared dispersed within the tunnels and developed into the larger mass of bone at 2-3 weeks (B). At 4 weeks, thick layers of bone were observed along the inner surface of the tunnels. In the central area of the tunnels, bone-marrow-like tissue that contained numerous adipocytes and young blood cells was clearly observed (C and D). Importantly, in both types of HCHAP, capillaries developed throughout the tunnels, eventually creating a structure similar to the Haversian system of bone remodeling. Bars indicate 100 m.
    Anchor for JumpAnchor for Jump
    +Fig. 4-A:Figure 4 Schematic representation of geometry-directed chondrogenesis and osteogenesis in the honeycomb hydroxyapatite with larger (A) and smaller (B) tunnels, which are applied as bone morphogenetic protein (BMP) carriers. In the smaller tunnels (90-110 m in diameter), cartilage was formed followed by bone formation, similar to the process of endochondral ossification. In contrast, in the larger tunnels (350 m in diameter) bone was formed directly without detectable cartilage formation. Figure 4-A reprinted, with permission, from: Kuboki Y, Takita H, Mizuno M, Fujisawa F: Geometry of artificial extracellular matrices: a new paradigm from dental tissue engineering. Dentistry in Japan. 2001;37:101.
    Anchor for JumpAnchor for Jump
    +Fig. 4-B:Figure 4 Schematic representation of geometry-directed chondrogenesis and osteogenesis in the honeycomb hydroxyapatite with larger (A) and smaller (B) tunnels, which are applied as bone morphogenetic protein (BMP) carriers. In the smaller tunnels (90-110 m in diameter), cartilage was formed followed by bone formation, similar to the process of endochondral ossification. In contrast, in the larger tunnels (350 m in diameter) bone was formed directly without detectable cartilage formation. Figure 4-A reprinted, with permission, from: Kuboki Y, Takita H, Mizuno M, Fujisawa F: Geometry of artificial extracellular matrices: a new paradigm from dental tissue engineering. Dentistry in Japan. 2001;37:101.
    Anchor for JumpAnchor for Jump
    +Fig. 5-A:Figure 5 Alkaline phosphatase activity (A) and osteocalcin contents (B) of implants of bone morphogenetic protein combined with porous blocks of hydroxyapatite of different pore sizes.
    Anchor for JumpAnchor for Jump
    +Fig. 5-B:Figure 5 Alkaline phosphatase activity (A) and osteocalcin contents (B) of implants of bone morphogenetic protein combined with porous blocks of hydroxyapatite of different pore sizes.
    Anchor for JumpAnchor for Jump
    +Fig. 6:Figure 6 Microphotographs of cross sections of bone morphogenetic protein (BMP)-combined porous blocks of hydroxyapatite 4 weeks after implantation. Pore sizes are 106-212 m in (A) and (B), 212-300 m in (C) and (D), 300-400 m in (E) and (F), 400-500 m in (G) and (H), and 500-600 m in (I) and (J). Original magnifications of photographs are 75 in the right lane and 150 in the left lane. Bars in the lower right corners of (I) and (J) indicate 100 m. Pores are shown as multiple round structures in all photographs (indicated by PO in A, for example). The spaces other than the pores (indicated by HAP in D, for example) have been occupied by hydroxyapatite. Bone formation was observed from the inner surface of almost all the pores (indicated by BO in F, for example) smaller than 500 m. On the other hand, bone not only formed along the inner surface of the pore but occupied the inner space (indicated by an arrow in I) in 500-600-m pores. Reprinted, with permission, from: Tsuruga E, Takita H, Itoh H, Wakisaka Y, Kuboki Y. Pore size of porous hydroxyapatite as the cell-substratum controls BMP-induced osteogenesis. J Biochem. 1997:121:317-24.
    Anchor for JumpAnchor for Jump
    +Fig. 7:Classification of geometry in extracellular matrices. Reprinted, with permission, from: Kuboki Y, Takita H, Mizuno M, Fujisawa F: Geometry of artificial extracellular matrices: a new paradigm from dental tissue engineering. Dentistry in Japan. 2001;37:101.
    Anchor for JumpAnchor for Jump  TABLE 1 Taxonomy of bone morphogenetic protein (BMP) carriers
    CarrierAbbreviationReference(s)
    Bone-inducing
    Porous particles of hydroxyapatitePPHAP2,4
    Porous blocks of hydroxyapatitePBHAP3
    Laser-perforated collagen membraneLPM14
    Cartilage-inducing
    Fibrous glass membraneFGM2,10
    Bone and cartilage-inducing
    Insoluble bone matrixIBM12
    Fibrous collagen membraneFCM5,11
    Collagen beadsCB16,17
    Fibrous mass of new bio-glassCPSA13
    Titanium meshTM18
    Honeycomb hydroxyapatiteHCHAP12
    Anchor for JumpAnchor for Jump  TABLE 2 Functions and properties of extracellular matrices
    PropertiesFunctions
    PhysicalMechanical support for cells, tissues, organs, and body
    ChemicalSupport of cells and molecules through surface charges; supply and deposition of various molecules
    BiochemicalSpecific interactions among adhesion molecules, signaling molecules (cytokines), and cell surface receptors
    GeometricalDirecting growth and differentiation of cells and tissues
    Kuboki Y, Yamaguchi H, Yokoyama A, Murata M, Takita H, Tazaki M, Mizuno M, Hasegawa T, Iida S, Shigenobu K, Fujisawa R, Kawamura M, Atsuta T, Matsumoto A, Kato H, Zhou H-Y, Ono I, Takeshita N, Nagai N. The bone-biomaterials interface. Osteogenesis induced by BMP-coated biomaterials. Biochemical principles of bone reconstruction in dentistry. In: The Bone-Biomaterial Interface, University of Toronto Press, Toronto, p 127-38, 1991 
     
    KubokiY, Saito T, Murata M, Takita H, Mizuno M, Inoue M, Nagai N,Poole AR. Two distinctive BMP-carriers induce zonal chondrogenesis and membranous ossification, respectively; geometrical factors of matrices for cell-differentiation. Connect Tissue Res,1995;32: 219-26. 32219  1995  [PubMed]
     
    TsurugaE, Takita H, Itoh H, Wakisaka Y,Kuboki Y. Pore size of porous hydroxyapatite as the cell-substratum controls BMP-induced osteogenesis. . J Biochem,1997;121: 317-24. 121317  1997  [PubMed]
     
    KubokiY, Takita H, Kobayashi D, Tsuruga E, Inoue M, Murata M, Nagai N, Dohi Y,Ohgushi H. BMP-induced osteogenesis on the surface of hydroxyapatite with geometrically feasible and nonfeasible structures: topology of osteogenesis. J Biomed Mater Res,1998;39: 190-9. 39190  1998  [PubMed]
     
    KubokiY, Sasaki M, Saito A, Takita H,Kato H. Regeneration of periodontal ligament and cementum by BMP-applied tissue engineering. Eur J Oral Sci,1998;106 Suppl 1: 197-203. 106 Suppl 1197  1998  [PubMed]
     
    UristMR. Bone: formation by autoinduction. Science,1965;150: 893-9. 150893  1965  [PubMed]
     
    ReddiAH. Cell biology and biochemistry of endochondral bone development. Coll Relat Res,1981;1: 209-26. 1209  1981  [PubMed]
     
    WozneyJM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, Hewick RM,Wang EA. Novel regulators of bone formation: molecular clones and activities. Science,242: 1528-34. 1988;2421528  1988  [PubMed]
     
    SasanoY, Ohtani E, Narita K, Kagayama M, Murata M, Saito T, Shigenobu K, Takita H,, Mizuno M,Kuboki Y. BMPs induce direct bone formation in ectopic sites independent of the endochondral ossification in vivo. Anat Rec,1993;236: 373-80. 236373  1993  [PubMed]
     
    SasanoY, Mizoguchi I, Takahashi I, Kagayama M, Saito T,Kuboki Y. BMPs induce endochondral ossification in rats when implanted ectopically within a carrier made of fibrous glass membrane. Anat Rec,1997;247: 472-8. 247472  1997  [PubMed]
     
    KobayashiD, Takita H, Mizuno M, Totsuka Y,Kuboki Y. Time-dependent expression of bone sialoprotein fragments in osteogenesis induced by bone morphogenetic protein. J Biochem. ,1996;119: 475-81. 119475  1996  [PubMed]
     
    JinQM, Takita H, Kohgo T, Atsumi K, Itoh H,Kuboki Y. Effects of geometry of hydroxyapatite as a cell substratum in BMP-induced ectopic bone formation. J Biomed Mater Res,2000;51: 491-9. 51491  2000  [PubMed]
     
    MahmoodJ, Takita H, Ojima Y, Kobayashi M, Kohgo T,Kuboki Y. Geometric effect of matrix upon cell differentiation: BMP-induced osteogenesis using a new bioglass with a feasible structure. J Biochem,2001;129: 163-71. 129163  2001  [PubMed]
     
    KikuchiM, Takita H, Nakayama Y, Matsuda T,Kuboki Y. Laser-perforated collagen membrane: pore size-dependent bone induction of a new BMP carrier. J Hard Tissue Biol,9: 2000;in press9  2000 
     
    MurataM, Inoue M, Arisue M, Kuboki Y,Nagai N. Carrier-dependency of cellular differentiation induced by bone morphogenetic protein in ectopic sites. Int J Oral Maxillofac Surg,1998;27: 391-6. 27391  1998  [PubMed]
     
    YokoyamaA. Experimental study of bone morphogenetic protein-purification and application for bony defect. Hokkaido J Oral Biology,1990;10: 151-84. 10151  1990 
     
    MizunoM, Kasagi T,Kuboki Y. Cross-linked collagen gel shares as a useful carrier for cell culture of MC3T3-E1 clonal osteogenic cells. Jpn J Oral Biol,1988;30: 855-853. 30855  1988 
     
    KubokiY, Takita H, Tsuruga E, Ono M,Jansen JA. Rationale for hydroxyapatite-coated titanium-mesh as an effective carrier for BMP. Dent Res,1988;77: 263. 77263  1988 
     
    ReddiAH,Huggins CB. Influence of geometry of transplanted tooth and bone on transformation of fibroblast. Proc Soc Exp Biol Med. ,1973;143: 634-7. 143634  1973  [PubMed]
     
    SampathTK,Reddi AH. Importance of geometry of the extracellular matrix in endochondral bone differentiation. J Cell Biol,1984;98: 2192-7. 982192  1984  [PubMed]
     
    van EedenSP,Ripamonti U. Bone differentiation in porous hydroxyapatite in baboons is regulated by the geometry of the substratum: implications for reconstructive craniofacial surgery. Plast Reconstr Surg,1994;959-66. 959  1994 
     
    TakitaH, Tsuruga E, Ono I,Kuboki Y. Enhancement by bFGF of osteogenesis induced by rhBMP-2 in rats. Eur J Oral Sci,1997;105: 588-92. 105588  1997  [PubMed]
     
    Yeh L-C,Lee JC. Osteogenic protein-1 increases gene expression of vascular endothelial growth factor in primary cultures of fetal rat calvaria cells. Mol Cell Endocrinol,1999;153: 113-24. 153113  1999  [PubMed]
     
    ParfittAM. The mechanism of coupling: a role for the vasculature. Bone,2000;26: 319-32. 26319  2000  [PubMed]
     
    TruetaJ. The role of the vessels in osteogenesis. J Bone Joint Surg Br,1993;45: 402-18. 45402  1993 
     
    OnoI, Ohura T, Murata M, Yamaguchi H, Ohnuma Y,Kuboki Y. A study on bone induction in hydroxyapatite combined with bone morphogenetic protein. Plast Reconst Surg,1992;90: 870-9. 90870  1992  [PubMed]
     
    OnoI, Gunji H, Kaneko F, Saito T,Kuboki Y. Efficacy of hydroxyapatite ceramic as a carrier for recombinant human bone morphogenetic protein. . J Craniofac Surg,1995;6: 238-44. 6238  1995  [PubMed]
     
    BassettAC,Herrmann I. Influence of oxygen concentration and mechanical factors on differentiation of connective tissues in vitro. Nature,1961;190: 4660-1. 1904660  1961 
     
    Kikuchi H, Kikuchi Y, Kuboki Y. Micropit effect on the growth of osteoblast-like cells studied using microfabricated pit arrays. Presented at the third annual meeting of the Japanese Tissue Engineering Society, Hiroshima, 2000 
     
    RipamontiU, Crooks J,Kirkbride AN. Sintered porous hydroxyapatites with intrinsic osteoinductive activity: geometric induction of bone formation. S Afr Sci,1999;95: 335-43. 95335  1999 
     
    den BraberET, de Ruijter JE, Ginsel LA, von Recum AF,Jansen JA. Orientation of ECM protein deposition, fibroblast cytoskeleton, and attachment complex components on silicone microgrooved surfaces. J Biomed Mater Res,1998;40: 291-300. 40291  1998  [PubMed]
     
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