Stem Cell Technology and Bioceramics: From Cell to Gene Engineering

Hajime Ohgushi,1 Arnold I. Caplan2

1

Department of Orthopedics, Nara Medical University, Kashihara City, Nara 634-8522, Japan Skeletal Research Center, Department of Biology, Case Western Reserve University, Cleveland, Ohio 44106

Received 14 December 1998; revised 16 April 1999; accepted 23 April 1999

Abstract: Mesenchymal stem cells reside in bone marrow and, when these cells are incorporated into porous ceramics, the composites exhibit osteo-chondrogenic phenotypic expression in ectopic (subcutaneous and intramuscular) or orthotopic sites. The expressional cascade is dependent upon the material properties of the delivery vehicle. Bioactive ceramics provide a suitable substrate for the attachment of the cells. This is followed by osteogenic differentiation directly on the surface of the ceramic, which results in bone bonding. Nonbioactive materials show neither surface-dependent cell differentiation nor bone bonding. The number of mesenchymal stem cells in fresh adult bone marrow is small, about one per one-hundredthousand nucleated cells, and decreases with donor age. In vitro cell culture technology can be used to mitotically expand these cells without the loss of their developmental potency regardless of donor age. The implanted composite of porous ceramic and culture-expanded mesenchymal stem cells exhibits in vivo osteo-chondrogenic differentiation. In certain culture conditions, these stem cells differentiate into osteoblasts, which make bone matrix on the ceramic surface. Such in vitro prefabricated bone within the ceramic provides immediate new bone-forming capability after in vivo implantation. Prior to loading of the cultured, marrowderived mesenchymal stem cells into the porous ceramics, exogenous genes can be introduced into these cells in culture. Combining in vitro manipulated mesenchymal stem cells with porous ceramics can be expected to effect sufcient new bone-forming capability, which can thereby provide tissue engineering approaches to patients with skeletal defects in order to regenerate skeletal tissues. 1999 John Wiley & Sons, Inc. J Biomed Mater Res (Appl Biomater) 48: 913927, 1999 Keywords: stem cell; ceramics; bone marrow; tissue culture; osteogenesis

INTRODUCTION When materials are inserted into host tissue, a rapid dilation of the local capillaries occurs with a subsequent increase in the permeability of their endothelial linings. As the permeability increases, water and larger molecules, including normal plasma proteins, move into the tissue. Shortly after these events, a series of cellular invasions takes place. The rst cells to appear at the site of implantation or injury are neutrophils, which function to phagocytose fragments of tissue or particles. This is followed by an inux of other cell types at the injury site, including eosinophils, monocytes, and macrophages. The macrophage also has phagocytic ability by virtue of its release of various bioactive molecules and can inuence the activity of many other cells, including lymphocytes, broblasts, osteoblasts, osteoclasts, and foreign body

Correspondence to: Hajime Ohgushi, Department of Orthopedics, Nara Medical University, Kashihara City, Nara 634-8522, Japan (e-mail: hajimeoh@naramed-u.ac.jp) Contract grant sponsors: Scientic Research Japan; National Institutes of Health 1999 John Wiley & Sons, Inc. CCC 0021-9304/99/060913-15

giant cells.1-3 These early events are referred to as the acute inammatory response and provide the cytokine and growth factor milieu, which results in the mitotic expansion of a number of mesenchymal cells, which then produce large amounts of extracellular matrix materials at the injury site. These newly synthesized macromolecules result in scar tissue and/or a brous membrane around the implanted materials. When metals or their alloys are implanted at host sites, such a brous membrane encapsulates the implants, with the thickness of the membrane proportional to the degree of metallic corrosion.4 However, in other in situ conditions or locations, these presumably same cells can foster an intrinsic repair response that covers the implanted materials with functional reparative tissue. For example, in bone defects, the mesenchymal progenitor cells, which expand around or within certain implanted materials, can undergo osteogenic differentiation, which results in functional regenerated bone tissue around the materials. When alumina (Al2O3) ceramics or titanium devices are implanted into the bone defects, the scar tissue/brous membrane formation is minimal, and some areas show regenerated bone tissue in contact with the im913

914

OHGUSHI AND CAPLAN

planted materials without other types of brous tissues intervening.5-11 However, the interface between the bone and the implants is not strong and the interface detaches easily upon shear and distraction loads. In this regard, when certain types of glass ceramics and calcium phosphate ceramics are implanted, chemical bonding is established between the regenerated bone tissue and the surface of the implant.12-20 This bone/ceramic interface is very strong and stable and, upon loading, breakage usually occurs inside the bone or ceramic, but not at the interface. Such interaction between bone and material (glass and calcium phosphate ceramics) is referred to as bone bonding. The bonding to bone was rst demonstrated by L. L. Hench for glasses within a certain compositional range. These glasses contained SiO2, Na2O, CaO, and P2O5 in specic proportions and were later termed bioactive glasses,12-14 with the bioactivity referring to the characteristic of an implant material that allows it to form a bond with living tissues. Though alumina ceramics can show bone apposition onto their surface without brous capsular formation, the bone cannot bond to the alumina surface and, thus, alumna is not bioactive. After discovery of the bonding property of the glasses and glass ceramics, hydroxyapatite (HA) ceramic and other types of calcium phosphate ceramics were found to show this bioactive property.19-30 These bioactive ceramics show surface changes, including dissolution and precipitation phenomena, which lead to carbonate-containing HA precipitation on the ceramic surface. In contrast, nonbioactive ceramics show neither the precipitation nor bone bonding.14,31-37 In fact, direct bone bonding has been reported via the apatite layer precipitated onto the bioactive ceramic.37 Since their discovery, many bioactive ceramics have been used for bone reconstruction surgery in clinical situations and have been reported to show excellent results. In addition, some nonceramic materials such as calcium carbonate crystals (aragonite and calcite)38-41 and organic polymers42-45 have been reported to show bone-bonding characteristics. Bone is formed by cells called osteoblasts, which arise from progenitor cells in a multistep lineage cascade.46,47 At bone defect sites, the osteoblast progenitors are recruited from tissues outside the bone and inside the bone (periosteum and bone marrow, respectively). That is, resected or injured bony sites induce the chemoattraction of multipotent mesenchymal progenitor cells, which originally reside in the marrow48,49 or periosteum, and, at the defect site, these cells become osteoblasts through a series of controlled differentiation steps. These progenitor cells, here referred to as mesenchymal stem cells (MSCs),50,51 have the capability to differentiate into various mesenchymal phenotypes, including osteogenic and chondrogenic cell types.52-54 As described, when metal or its alloy is implanted at the bone defect site, the implant is covered with a thick brous capsule,4 which precludes osteogenic differentiation on the implant surface. In contrast, alumina ceramics or titanium surfaces allow osteogenic differentiation near the implant surface, with bioactive ceramics exhibiting this differentiation directly on their surfaces.55-57 Therefore, a key issue in successful implanta-

tion is the response and differentiation of MSCs around the implanted materials.28,29,58-61 To evaluate a particular materials performance as it relates to bone regeneration, the material can be implanted into orthotopic sites, usually into osteotomies of long bones. In these situations, implanted material in close proximity to the bone surface exhibits a better osteogenic response than the implanted material distal to the surface. Such precise implantation is highly demanding, especially in small animal models, and, thus, requires sufcient numbers that provide statistically signicant results. With this stringency in mind, a less demanding experimental protocol was developed to show bone formation around the implants by grafting the marrow MSC/material composites into subcutaneous sites.24-30,5560,62-65 The method is simple and rapid, with the insertion of the composite materials into subcutaneous pouches, which are created by blunt dissection with scissors. These cell/material composites exhibit osteogenic differentiation and bone deposition in and around the materials and allow both quantitative and qualitative evaluation of various materials with regard to osteogenic response.28,29,58-60,64,65 Both de novo bone and cartilage formation derived from donor marrow MSCs have been reported; thus, this approach provides an assay for the multipotential character of the donor cells. Some years ago, we rst initiated studies with fresh, dispersed whole marrow cell preparations.24 Although the number of MSCs in the fresh marrow cells is small, the use of high numbers of fresh marrow cells in the implanted composites contributed to the de novo bone and cartilage formation.24-30 Following these initial studies, the technology for cell culture was developed to isolate and mitotically expand MSCs, while maintaining their capability to differentiate in vivo into osteogenic and chondrogenic tissues.62-70 Furthermore, conditions have been devised to induce these cultured MSCs to differentiate into osteogenic and chondrogenic phenotypes in vitro.71-79 When combined with bioactive ceramics, these cultured, multipotent mesenchymal progenitor cells or isolated, mature differentiated cells can survive the process of in vivo implantation and can contribute to skeletal tissue repair.80,81 We focus here on the interaction between MSCs and various biomaterials and emphasize the importance of the surface of the bioactive ceramic for MSC differentiation. This differentiation capacity is the key for successful tissue engineered skeletal tissue repair/regeneration with cell/material composite grafts.

MATERIALS PERFORMANCE
Bioactive Materials (Calcium Phosphate and Glass Ceramics)

By analyzing composites of fresh, dispersed rat marrow cells and porous HA ceramic transplanted into heterotopic subcutaneous sites, consistent de novo bone formation has been observed.26-30,58-60 Interestingly, the newly formed bone is usually found inside the internal pore regions and not outside

STEM CELL TECHNOLOGY AND BIOCERAMICS

915

on the surface of the ceramic. This suggests the importance of cell retention inside the pore area,26-28,62 and the disruptive capabilities of the initial acute inammatory response, which can potentially strip cells off of the outer surface of the ceramic. Sequential (temporal) analysis26,27,59,60 of the bone formation indicates that: (1) only a few mesenchymal progenitor cells from whole dispersed marrow attach on the ceramic pore surface; (2) the attached cells proliferate on these ceramic surfaces; (3) the cells form a continuous cell layer on the surface of the ceramic within the pores; (4) cell differentiation of these progenitor cells results in the appearance of a continuous layer of osteoblasts on the pore surface; (5) the osteoblasts fabricate immature primary bone onto the surface; and (6) the primary bone becomes mature and forms fully mineralized bone. Before the attachment of these cells, the ceramic shows the surface changes, including dissolution and precipitation phenomena, which lead to the precipitation of a carbonate-containing HA layer on its surface.31-34 It is well known that HA adsorbs many proteins and other macromolecules, and, thus, during formation of the HA layer on the ceramics, other biological molecules also integrate into the layer. These surface changes favor cell attachment and subsequent cell differentiation. Though the nature of the biological molecules to evoke the attachment and differentiation are not certain, cell-binding proteins having the RGD (arg-gly-asp) sequence probably have some role. In this regard, the appearance of osteopontin mRNA expression in the implanted marrow/ceramic composite has been documented,82 and exogenously added bronectin or laminin promotes MSC attachment and osteoblastic differentiation in the composites.62,63 Both osteopontin and bronectin contain an RGD sequence. Others also reported the importance of osteopontin83,84 and bronectin85 molecules in the bone/material interface. These molecules on the ceramic surface play a role in attracting MSCs and, thus, contribute to the osteoblastic differentiation process. These differentiated osteoblasts fabricate bone directly on the ceramic surface, and this bone tissue is composed of both organic and inorganic matrix phases with carbonate-containing HA. A similar crystal structure can be formed on the ceramic surface by the dissolution and precipitation phenomena; both ceramic and natural bone crystals unite and nally establish chemical bonding between the bone and the ceramic. The developmental cascade, which results in functional bone bonding, is initiated by the attachment of MSCs in the dispersed marrow cell populations followed by osteoblastic differentiation and is schematically represented in Figure 1. MSC attachment and subsequent differentiation are depicted in Figure 1(a), which shows the attachment of broblastic cells (stem cells), which develop into small round cells stained weakly with an osteocalcin antibody (preosteoblasts). Further down are clusters of larger round cells that are strongly stained with osteocalcin antibody; these osteoblasts fabricate a thin layer of primary bone onto the ceramic surface. The primary bone is partially mineralized osteoid, and later this tissue becomes fully mineralized bone, and

bone bonding becomes established. As shown in Figure 1(b), mineral content (calcium) in the thin primary bone is low, which is an indication of osteoid formation, but the calcium and phosphorus content in the area of thick bone is comparable to those of the ceramic [Fig. 1(d)]. Importantly, these high levels of mineral are found across the bone/HA ceramic interface, and there is no intervening brous tissue at the interface, which, again, implies bone bonding. After fully mineralized bone formation, appositional bone formation follows; thus, invariably, bone formation always starts on the surface of the HA ceramic and proceeds toward the center of the pore. As shown in Figure 1(c), tetracycline injected at 5 weeks is seen close to the ceramic surface, whereas calcein injected at 6 weeks is seen away from the ceramic surface. The cascade is called bonding osteogenesis 21 and indicates the importance of a bioactive ceramic surface to support the osteogenic differentiation of marrow MSCs. By analyzing the cell/material composite graft, other bioactive materials such as glass ceramics (apatite wollastonite-containing glass ceramics), tricalcium phosphate (TCP) and biphasic HA/TCP ceramics have been found to exhibit the same cascade of bonding osteogenesis 24,25,28-30,86 and to show the same material performance relative to the cell differentiation sequence.
Bioactive Material (Calcium Carbonate)

Marine coral has been used as a bone graft substitute in Europe, especially in France.38,39 The 3-dimensional macroporous structure mimics natural cancellous bone and facilitates tissue and vascular invasion into the pore areas after implantation of the coral. Compositional analysis of the coral showed the crystalline form of calcium carbonate to be aragonite with a trace of organic elements.38 Experimental and clinical data show excellent vascular invasion, biocompatibility, and osteoconductivity of the coral when used as a bone graft substitute. Histologic analysis after implantation of the coral into a bone defect showed bone adaptation to the coral surface; the crystalline form of calcium carbonate as calcite also showed strong bone adaptation to the calcite surface.40 These observations imply a strong afnity of bone tissue to the coral surface (calcium carbonate). Analysis of implants of composites consisting of dispersed whole marrow cells and coral showed that the surface of the coral (calcium carbonate) also supported osteogenic differentiation of marrow MSCs as evidenced by the bone-forming pattern (bonding osteogenesis).29,30,41 As shown in Figure 2, continuous high levels of calcium were observed across the interface of the newly formed bone and coral. These observations indicate the bioactive nature of coral, although the composition of the coral is different from other bioactive ceramics. The bioactive glass and calcium phosphate ceramics contain calcium and phosphorus; in addition, the bioactive glass contains silica, which may have an important role for the bioactivity.12-14 Though coral contains calcium, it contains neither phosphorus nor silica. The bioactive glass and calcium phosphate ceramics show surface changes, including

916

OHGUSHI AND CAPLAN

STEM CELL TECHNOLOGY AND BIOCERAMICS

917

Figure 2. Back-scattered electron image photograph of calcium carbonate (CC) loaded with fresh dispersed marrow cells 8 weeks after implantation. Intervening brous tissue (black area) is not observed between de novo bone (gray area) and calcium carbonate (CC). The panel on the right shows a higher magnication of the rectangular area seen on the left. Simultaneous line analysis of calcium (Ca), phosphorus (P), and magnesium (Mg) are shown as light blue lines from the electron beam pathway as indicated by the red line. A continuous high level of calcium is observed at the interface between CC and bone, while the level of phosphorus in CC is negligible.

dissolution and precipitation phenomena, which lead to the formation of carbonate-containing HA on their surface. Interestingly, the apatite layer can be formed on the bioactive ceramic surface when it is soaked in a simulated physiologic solution that mimics the ion concentrations in body uids.31-36 Based on these results, coral and porous HA (both having the same macro porosity and pore size) were soaked in a solution having ionic concentrations of 142 mM Na, 5.0 mM K, 1.5 mM Mg2, 2.3 mM Ca2, 148.8 mM Cl, 4.2 3 mM HCO 3 , and 1.0 mM PO4 at 37C, pH 7.7. Both HA and coral showed rapid carbonated apatite formation after a lag

time of 1 6 h, respectively, which conrms the bioactive property of coral (calcium carbonate).33,34 However, the in vivo degradation property of coral is different from that of HA. Hydroxyapatite shows essentially no degradation even 1 year after implantation. The marrow/HA ceramic composites showed almost no macroscopic degradation of the ceramic, and the de novo bone inside the porous areas of HA exists for a long time period (described later); others also reported that the rate of degradation of HA is very slow.23 In contrast, implanted coral, with or without marrow, showed almost complete degradation after 6 months. Therefore, coral is both

Figure 1. Marrow cell/hydroxyapatite ceramic composite implantation at subcutaneous sites. As schematically shown in the upper left corner, when whole marrow cells are combined with ceramic hydroxyapatite (brown area), de novo bone formation (gray area) together with clusters of osteoblasts (round cells) can be observed. Red boxed areas are the focus of (a) to (d). (a) Hisotologic view of preosteoblasts, presumably derived from MSCs, on the surface of the ceramic. The preosteoblasts exhibit weak immuno-signals for osteocalcin, while active osteoblasts exhibit strong osteocalcin signals, as visualized by the brown color of the immunostain with anti-rat osteocalcin antibody. The osteoblasts fabricate new bone directly on the surface of the ceramic. (b1) Initial bone formed on the ceramic surface observed by scanning electron microscopy. (b2) Electron probe microanalysis for calcium was performed as indicated by the dotted line in b1. The calcium contents of the ceramic, bone, and cell areas are shown in the yellow column from left to right. The calcium content of the initial bone area is about 1/3 that of the ceramic area. The low content of calcium in the bone indicates that the bone is not fully mineralized. (c1) Light micrograph of an undecalcied section of the bone formed on the ceramic surface. The white area shows the newly formed bone, and the brown area is the ceramic. (c2) The same section as in c1, but viewed with uoromicroscopy. Yellow tetracycline (Tet) and green calcein (Cal) were administrated 5 and 6 weeks after implantation, respectively. The uoromicroscopy shows that bone rst formed on the ceramic surface and then proceeded to be fabricated away from this surface. (d) Back-scattered electron image, which displays no intervening soft tissue (black area) between bone (gray area) and ceramic (white area). Simultaneous line analysis of phosphorus (P) and calcium (Ca) are derived from the electron beam pathway as indicated by the dotted line. The calcium content of the bone is high (about 75% of that of the ceramic), which indicates fully mineralized bone, compared to the low content of calcium in the initial bone on the ceramic surface (b). (a), (b), and (c), (d) are modied from gures in Refs. 60 and 26, respectively.

918

OHGUSHI AND CAPLAN

Figure 3. Eight weeks after subcutaneous implantation of alumina ceramic loaded with fresh dispersed marrow cells. The left panel shows an undecalcied section under light microscopy (red bar 25 m). The de novo bone is attached (arrows) to the surface of the alumina ceramic (AL). The right panel shows the same section under uoromicroscopy. Tetracycline (yellow color) and calcein (green color) were administrated 5 and 6 weeks after implantation, respectively. The uoromicroscopy reveals that bone formation was initiated away from the surface of the ceramic, probably around the Starting point indicated in the left gure. Modied from gures contained in Ref. 55.

resorbable and bioactive, and this may be of considerable benet in certain clinical situations.
Nonbioactive Materials (Alumina and Titanium)

In contrast to the bone formation pattern in the implants of marrow mesenchymal progenitor cell/bioactive material composites, bone formation always starts away from the surface in the marrow cell/nonbioactive materials. Usually an intervening brous layer covers and encapsulates the nonbioactive materials, and the de novo bone does not directly contact the material. However, in the composites of marrow cells and some nonbioactive materials, such as alumina ceramics and titanium implants, a number of areas showed bone apposition without intervening brous tissue55-57; these materials have been categorized as bioinert materials. As shown in the Figure 3, the marrow cell/alumina composite shows contact of newly formed bone to the material surface; however, urochrome labeling demonstrated that bone formation started away from the material surface.55 At the areas of bone/material contact, newly formed bone formed towards the material surface and replaced the intervening brous tissue; bone bonding did not occur in this situation. It is also known that alumina does not show surface changes characteristic of apatite formation on the implant surface. Therefore, alumina is not classied as bioactive. Implanted marrow cell/titanium composites exhibit results comparable to those for marrow cell/alumina composites, with some areas showing bone contact with the titanium, although bone formation started away from the titanium surface.56 However, titanium is somewhat different from alumina ceramics, because titanium is a reactive metal on which an oxide can spontaneously form; this oxide has been shown to react with mineral ions

and fabricate a calcium phosphate layer.32,87-89 Therefore, there is the possibility that titanium can function as a bioactive material under some conditions. These observations of marrow/material composites showed that the implantation of composites is a useful tool to distinguish bioactive materials from nonbioactive materials. The current experimental results indicate that, regardless of the fabrication methods, if the materials exhibit surface changes that lead to carbonate-containing HA formation, the materials have the property of bone bonding and, thus, are bioactive. Importantly, the bone bonding process occurs after attachment of MSCs onto the surface and as a consequence of the subsequent osteogenic differentiation on the newly formed HA crystals. This differentiation process is also dependent on the pore dimensions of the material used for making the composites with marrow cells. For fresh dispersed marrow cells and for cultureexpanded MSCs as the cell sources, a pore diameter from 200400 m is suitable, and for the osteogenic matrix coating, which is described later, the diameter around 500 m is suitable.26-30, 66-68 The pore interconnection frequently is also a critical factor in the osteogenic differentiation process of marrow cells and MSCs in the porous materials. Complete interconnection of the pores allows the cells access to deeper regions in the materials and also ensures complete vascular penetration after in vivo implantation. As pointed out previously, there is an obligatory interaction between vasculature and the osteogenic process.24,46,54,62-64 MANIPULATION OF MESENCHYMAL STEM CELLS
Osteogenic and Chondrogenic Differentiation

The composite of fresh dispersed whole bone marrow cells and porous calcium phosphate ceramics exhibits osteo-chon-

STEM CELL TECHNOLOGY AND BIOCERAMICS

919

Figure 4. Schematic representation of the temporal events of bone/cartilage formation of various composites of porous HA ceramic and fresh marrow cells or MSCs after subcutaneous implantation. (A) Composite containing fresh dispersed marrow cells; (B) composite containing culture- expanded MSCs; (C) composite containing culture-expanded MSCs allowed to differentiate in vitro into osteoblasts, which make bone matrix on the ceramic. The bone fabricated on the ceramic in vitro exhibits rapid (1 week) and massive bone formation across the in vitro/in vivo transition.

drogenic differentiation (appearance of osteoblasts and chondrocytes) when implanted at subcutaneous or intramuscular sites.24-30 Although the number of fresh marrow cells used in such implants is very large, only a small number of MSCs reside in this marrow preparation, and these mitotically expand at the site and then differentiate either into osteoblasts or chondroblasts. Alternatively, marrow cells can be seeded into culture, where MSCs attach and mitotically expand.66-74 For example, from one rat there is sufcient marrow to directly establish only 23 composite grafts, which exhibit in vivo osteo-chondrogenic potential. However, after in vitro expansion, there are sufcient cells (rst passage cells) to establish 20 30 composites, which give a substantial osteogenic response.67 Thus, the number of stem cells can be amplied about 10 times with only a single passage in culture.73 We

initially used dispersed rat marrow cells for making the osteogenic composites24; in vitro expanded MSCs are now employed because more rapid, uniform bone formation is observed.62,63 When fresh, whole marrow cells [Fig. 4(A)] or culture-expanded MSCs [Fig. 4(B)] were combined with porous ceramics, the composites exhibited in vivo osteogenic potential, with those containing cultured MSCs observed to form bone much earlier.69 Importantly, cultured MSCs can be passaged over 30 population doublings in vitro, which represents an over one-billion-fold expansion, without loss of testable osteogenic potential.70,73 These observations document the usefulness of the in vitro culture expansion technique with retention of osteo-chondrogenic potential as observed after in vivo implantation.66-71 Importantly, in the presence of inductive agents such as

920

OHGUSHI AND CAPLAN

dexamethasone, TGF-, BMP-2, -4, or -7, or growth hormone, expanded stem cells are forced into the osteogenic72,73,77-79 or the chondrogenic75 lineages in vitro. These newly differentiated cells not only exhibit their specic phenotypic characteristics, but also fabricate a voluminous and specic extracellular matrix. Especially in the presence of dexamethasone and -glycerophosphate, the stem cells can differentiate into osteoblasts and produce mineralized bone matrix.77-79,90-92 Detailed analysis of the cells and their in vitro fabricated matrix showed these cells to be osteoblasts, as evidenced by the appearance of osteoblastic markers at the levels of protein and gene expression.73,77,78,93 X-ray diffraction and Fouriertransform infrared spectroscopy further revealed that the mineralization was composed of carbonate-containing HA comparable to natural bone mineral.78 Therefore, by culturing the marrow MSCs, their number can be expanded without loss of their in vivo osteogenic potential. These same cells can be manipulated into either bone-forming osteoblasts or cartilageforming chondroblasts under appropriate in vitro culture conditions.73,75,78,93 As described above, the composites of culture-expanded MSCs and porous ceramics can show in vivo bone formation at subcutaneous sites, and the results indicate the maintenance of cell function through in vitro culture expansion procedures [Fig. 4(B)]. In this regard, when MSCs were cultured in a porous framework on HA under conditions (dexamethasone) where the stem cells can differentiate into osteoblasts and fabricate bone matrix, the porous areas of the HA were covered with de novo bone and active osteoblasts79 [Fig. 4 (C)]. The bone matrix fabricated in vitro can be visualized by scanning electron microscopy, but is thin and difcult to observe by ordinary light microscopy. This in vitro composite exhibits high alkaline phosphatase activity and an increase in the bone matrix protein, osteocalcin, after subcutaneous implantation.68 One week after in vivo implantation of this in vitro composite, obvious bone formation could be observed in histologic sections by ordinary light microscopy [Fig. 4(C)].68,79 Such bone formation at one week after implantation has never been observed when composites of fresh marrow or cultured MSCs and porous ceramics were implanted [Figs. 4(A) and (B)]. Interestingly, levels of gene expression of alkaline phosphatase and osteocalcin were comparable to those of cancellous bone as early as 2 weeks after implantation.94 These observations indicate that either culture-expanded MSCs or culture-differentiated osteoblasts survive the in vitro/in vivo transition. These techniques have substantial clinical signicance, as described later.
Repair of Bone Defects

porous ceramics and implanted into rat80,95 or canine segmental bone defects,81 which by themselves cannot heal. Two months following implantation in orthotopic sites, the defects that received ceramics without marrow showed only a minimum amount of new bone, which arose from the cut end of the host bone [Fig. 5(A)]. In contrast, the composites of marrow and ceramic showed bony continuity brought about by regenerated bone tissue in and around the ceramics, which indicates the healing potential of the composites [Fig. 5(B)]. Based on these data, composites of aspirated autologous bone marrow and porous HA ceramics can be used for the treatment of patients having benign bone tumor excision or intraarticular compression fractures.96
Repair of Cartilage Defects

Repair of cartilage damage or defects is technically challenging, because cartilage tissue is relatively thin and avascular. Since it overlays subchondral bone tissue, damage to cartilage often is accompanied by damage to subchondral bone. Therefore, repair of both tissues must often be considered. Lastly, for a variety of reasons, the intrinsic healing potential of cartilage is very poor or nonexistent. In an attempt to provide regeneration of both cartilage and bone, cultured MSCs were implanted into massive osteochondral defects in the medial condyle of the distal femur of young adult rabbits. The MSCs uniformly formed chondrocytes, which served to resurface the condyle.97,98 In contrast, the cells at the bony base of the defect formed chondrocytes, which continued to rapidly progress along the chondrogenic lineage and become hypertrophic chondrocytes. These hypertrophic chondrocytes expired and attracted host vascular cells and resorbing cells. Host-derived MSCs were attracted and subsequently their descendants fabricated bone (Fig. 6). Thus, with one batch of cultured MSCs, the cartilaginous layer together with underlying subchondral bone can be regenerated to form normal joint architecture. Because MSCs are immature progenitor cells, they are capable of differential lineage progression, which depends on local cueing and the local environment. In this cartilage defect model, it appears that the entire population of implanted MSCs initially progresses to the chondrocyte phenotype. At this stage, mechanical and biological factors, presumably arising from the bone surrounding the defect and synovial space, appear to stimulate cells in the subchondral zone to differentiate further into hypertrophic chondrocytes. However, chondrocytes in the articular zone must be inhibited from such lineage progression, presumably by factors arising from the synovium, and are locked into the articular chondrocyte phenotype.97,98

When fresh marrow cells or culture-expanded MSCs are combined with porous ceramics, the composite exhibits osteogenic/chondrogenic activity in subcutaneous/intramuscular sites (heterotopic sites). The more important question is whether the composite can exhibit this healing potential in bone/cartilage defects (orthotopic sites). In this regard, either fresh marrow cells or cultured MSCs were combined with

FUTURE OF THE STEM CELL TECHNOLOGY

Osteogenic Matrix Coating on Biomaterials

Marrow MSCs can be expanded and manipulated in vitro and, importantly, these cells survive after in vivo implantation and subsequently differentiate into skeletal phenotypes. This

STEM CELL TECHNOLOGY AND BIOCERAMICS

921

Figure 5. Repair of nonunion rat femoral defect with implanted marrow cell/hydroxyapatite ceramic composite. (A) Schematic representation of the ceramic in the rat femoral defect (5 mm). (B) Decalcied histologic section of the femur 2 months after surgery (arrows indicate the bone/ceramic junction). The pores of the ceramic were lled with bone; a bony continuity was observed at both bone/ceramic junctions. Modied from gures in Ref. 95.

culture technique can be applied to marrow mesenchymal progenitor cells from various animal sources, including human. Older animals and aged patients have diminished rates of repair of skeletal tissues and decreased numbers of MSCs.99,100 In concert with these observations, fresh rat marrow/ceramic composites exhibited decreased bone-forming capability when the marrow was taken from aged rats, even when the composites were implanted into young recipients.99,101 These data imply that the decreased number of MSCs in aged animals accounts for the observed decreased rates of bone repair. The number of human MSCs in bone marrow likewise decreased from 1 per 10,000 marrow cells in newborn marrow to 1 per 2,000,000 for geriatric patients.99,102 Although the MSC number in marrow decreased with age, the number of MSCs can be expanded under culture conditions, where MSCs retain their ability to differentiate into osteogenic lineage regardless of the original donors age.73,99 Importantly, the culture-expanded MSCs from the marrow of very old patients can be expected to exhibit effective osteogenic differentiation. Thus, aged patients with skeletal defects can benet from tissue engineering approaches to regenerate skeletal tissue. Culture-expanded MSCs can be combined with bioactive ceramics and implanted66-71[Fig. 4(B)], or MSCs can be

induced to differentiate into active osteoblasts on bioactive ceramics in vitro77,79 and then be implanted68,94[Fig. 4(C)]. These techniques (one using cultured autologous progenitor cells and the other using progenitors that have formed differentiated cells) provide two novel tissue engineering strategies for repairing skeletal injuries or decits. Both techniques have different merits. Immature progenitor cells have the capability of progressing into various mesenchymal lineages and, thus, are useful for repairing tissues that have different architecture, such as in joints (cartilage tissue at the surface and cancellous bone beneath).53,97,98 The advantage of using in vitro differentiated or lineage-progressing cells is that the repair of the tissue should occur faster than with the use of progenitor cells. Importantly, in the culture conditions that stimulate MSCs into osteogenic differentiation72,73,77-79,93 the in vitro differentiated cells also generate bone matrix, which contains many biologic factors (BMPs, TGF-, and other growth factors or cytokines) that contribute to rapid healing. As an aside, the same MSCs that can form cartilage and/or bone can also form tendon or ligament, depending on the site of implantation and the properties of the delivery vehicle. For example, rabbit MSCs encased in a collagen gel that is contracted around a restorable suture can differentiate into tendon when placed into an Achilles tendon defect under

922

OHGUSHI AND CAPLAN

Figure 6. Cartilage repair with rabbit MSC implant. Diagram based on observations of the repair dynamics of a full-thickness defect in the medial condyle of the distal femur of young adult rabbits. The defect is lled with a type I collagen gel containing culture-expanded autologous marrow-derived or periosteum-derived MSCs[97]. The implanted MSCs differentiate into chondrocytes, probably due to the bony receptacle-derived bioactive molecules, perhaps TGF- or BMPs. The cells in contact with the bone and at the base further differentiate into hypertrophic chondrocytes, which expire and are replaced by vasculature. New bone then forms from host-derived MSCs brought into the defect by the vasculature. The chondrocytes at the articular surface do not become hypertrophic, perhaps due to synovial or mechanical factors. Eventually, the defect is lled with articular cartilage on the new bone, which is further remodeled to be indistinguishable from host bone.

load.53,103 Likewise, culture-expanded MSCs can be seeded onto a polymeric material, which can be used in an anterior cruciate ligament reconstruction.104 Thus, MSCs may also be used to bridge various skeletal tissues by allowing these undifferentiated progenitors to respond to local cueing at the implantation site. As shown in Figure 4(C), when cultured MSCs were seeded onto the surface of bioactive ceramics under conditions that induce the cells to differentiate into osteoblasts, in vitro bone (active osteoblasts with bone matrix) could be constructed on the ceramic. When implanted in vivo, this composite continued to fabricate new bone. Likewise, such in vitro differentiated cells on nonbioactive materials provide such materials with a covering of genuine bone tissue. On both bioactive and nonbioactive materials, HA crystals, biologic factors, and osteoblasts are all produced and derived from the donor cells. Thus, in vitro cultured bone on the surface of materials functions with bone bonding together with new bone-forming capability. The bonding capability is derived from HA crystals in the bone matrix, and the new bone-forming capability is attributed to osteoblasts and many biologic factors, including BMPs. Using this technique, we can alter the surface of nonbioactive materials through bioactive substances having osteogenic function.

Many osteoarthritic and rheumatoid arthritic patients need total joint replacements; these prosthetic devices have problems including loosening of the implants. To prevent loosening, the prostheses are fabricated with porous structures105,106 or coated with bioactive materials such as HA107-111 (Fig. 7). We propose a new concept to prevent such loosening, which is to coat joint prostheses with osteogenic cells or their precursors. As shown in Figure 7, fresh bone marrow would be collected from the patient, and the MSCs isolated, expanded in number in culture, and subsequently cultured on the surface of the prostheses under osteogenic conditions. The surface of the prostheses would be covered with bone (osteoblasts and bone matrix) derived from the patients own cells. This bone would possess the capability for bone bonding as well as new bone formation. Due to this biologic surface reconstruction, loosening can be avoided, while the postoperative rehabilitation program can be shortened due to early and secure bone formation around the implanted prosthesis. The concept, designated as osteogenic matrix coating on the prosthesis (Fig. 7), requires two invasive steps: one is the harvesting of whole marrow and the other is the surgery for total joint replacement. Fortunately, removing marrow by aspiration from the iliac crest is straightforward and only a few milliliters can provide sufcient MSCs for

STEM CELL TECHNOLOGY AND BIOCERAMICS

923

Figure 7. Schematic diagram of osteogenic matrix coating. To prevent loosening of a total joint replacement, the current approach is to apply porous coatings or HA on the stem of the protheses used for total hip joint replacements. To prevent loosening of these protheses, the patients bone marrow cells are rst cultured to increase the number of MSCs, and these cells are subsequently seeded onto the prostheses in the presence of osteogenic stimuli (dexamethasone (Dex) and -glycerophosphate) to stimulate the differentiation of osteoblasts, which coat the prostheses with bone matrix. The osteogenic matrix coating on the prostheses could be expected to secure the prostheses in vivo.

coating the ceramic or implant.112 This osteogenic matrix coating can be used for other skeletal reconstruction surgery, such as therapy for patients having bone tumor excisions, fractures with massive bone defects, and delayed/nonunion of fractured bone. For these patients, various biomaterials can be used for the therapy, after the materials are coated with bone matrix by osteoblasts derived from the MSCs of that patient. These engineered materials with their osteogenic matrix coating should be secure and provide rapid bone healing.
Gene Delivery System

Fresh dispersed whole marrow as well as in vitro cultureexpanded MSCs have been shown to exhibit osteoblastic and chondroblastic expression in porous bioactive ceramics after

in vivo implantation. These composites, containing either fresh whole marrow cells or culture-expanded MCSs, show osteoblastic activity for as long as a year, as conrmed by high alkaline phosphatase activity113 and mRNA expression of bone-specic osteocalcin. Interestingly, the mRNA expression is about 40% of that in normal rat cancellous bone, which indicates the persistent osteoblastic activity comparable to the normal bone milieu (Fig. 8).114 Histologic bone tissue together with osteoblasts and osteocytes was found only in the pore areas and not outside the ceramic area. Bone-resorbing osteoclasts together with regenerated marrow tissue were also detected in composites harvested at one year following implantation (Fig. 8). These observations indicate that new bone and marrow tissue can be fabricated inside the pore regions of the ceramics at ectopic (intramuscular or subcutaneous) sites, and, importantly, this tissue persists and exhibits cellular activity comparable to normal bone and marrow tissue. The persistent cellular activity suggests that the implanted MSCs initiate a differentiation cascade, which is perpetuated by host-derived cells; it is possible that some proportion of the MSCs persists as self-replicating progenitors, which contribute to the osteoblast or marrow stromal cell population. With the cell culture technology, the number of MSCs can be expanded and an exogenous gene can be introduced into the cells. Based on the persistence of osteogenic tissue, persistent gene expression should also be expected in the pores of the ceramics after in vivo implantation of the cultured stem cell/ceramic composites. Using myeloproliferative sarcoma virus (MPSV)-based retrovirus, the genes for -galactosidase (-Gal) or human Interleukin-3 (IL-3) have been inserted into the genome of human MSCs.115 The transduced MSCs were combined with porous bioactive ceramics and implanted at subcutaneous sites of nude or SCID mice, where osteogenesis by gene-marked osteoblasts and osteocytes was observed. Circulating human IL-3 was observed in the mice that received ceramics loaded with human MSCs transfected with IL-3-containing retroviral constructs. Thus, various gene insertion constructs can be used with MSCs in culture to introduce various exogenous genes into these cells. Other vectors such as adenovirus and liposome can be used for gene delivery into MSCs, although the retrovirus vector seems to be the most effective and consistent in ensuring persistent expression of the transduced gene. With this or other genetic engineering approaches, viable new tissue derived from mesenchymal progenitor cells having the exogenous gene can be fabricated inside the porous areas of ceramics at subcutaneous or intramuscular sites. This new tissue can produce functional proteins corresponding to the transferred gene; these de novo proteins can be incorporated into the new bone matrix, or they can be secreted into the circulatory system, which interfaces with the newly forming bone. Taken together with the ndings of persistence of the tissue formed from the MSC/ceramic composites, new gene therapy may be possible for patients lacking specic protein production due to the dysfunction of a specic gene. The patients can be treated by subcutaneous or intramuscular

924

OHGUSHI AND CAPLAN

Figure 8. One year after subcutaneous implantation of marrow cell/HA ceramic composite. The left panel shows the decalcied section of the composite, where almost all pore areas are lled with bone. Regenerated bone marrow also can be seen in some pore areas (black bar 200 m). The right panel shows a Northern blot analysis of mRNA for osteocalcin. Total RNA was extracted from cancellous bone, bone marrow, marrow/ceramic composite, and ceramic without (w/o) marrow. These denatured RNA samples were electrophoresed, stained with ethidium bromide (A), and transferred to a nylon membrane. Autoradiogram of Northern blot probed with 32P-labeled cDNA is seen in B. Ribosomal RNAs of 28S and 18S are indicated as 28S and 18S, respectively. Only the RNA from bone and the marrow/ceramic composite exhibit a signal for osteocalcin mRNA. Modied from gures in Ref. 114.

implantation of a composite of porous bioactive ceramics, and the patients MSCs transfected with the normal gene. Alternatively, patients can be subjected to marrow ablation by chemotherapy followed by bone marrow transplantation with supplemental culture-expanded MSCs transfected with the normal gene.116 Such xed MSCs home back to the bone marrow and serve as a continuous supply of progenitor cells for a variety of mesenchymal tissues. In the case of Osteogenesis Imperfecta, this approach could potentially be curative. These and other therapeutic approaches provide an exciting new dimension of cell-based therapy.117-119
We thank our colleagues at Nara Medical University and Case Western Reserve University. We especially thank Drs. Motoaki Okumura, Takafumi Yoshikawa, Keisuke Inoue, Siro Tabata, Yoshiko Dohi, Shigeyuki Wakitani, Randell Young, Scott P. Bruder, David A. Carrino, Stephen E. Haynesworth, David J. Fink, James A. Allay, and Stanton L. Gerson. We also thank Dr. Paul Ducheyne (Univ. of Pennsylvania) for collaboration regarding titanium and calcium carbonate and Dr. Edwin C. Shors (Interpore International, CA) for providing ceramics. A special note of gratitude is extended to Professors Susumu Tamai and Victor M. Goldberg for providing the stimulating intellectual atmosphere in which these studies took place.

6.

7.

8.

9.

10.

11.

12.

13. 14.

REFERENCES
1. Black J. The inammatory process. In: Black J, editor. Biological performance of materials. New York: Marcel Dekker; 1992. p 125145. 2. Ziats NP, Miller KM, Anderson JM. In vitro and in vivo interactions of cells with biomaterials. Biomater 1988;9:513. 3. Anderson JM. Inammatory response to implants. ASAIOTrans 1988;34:101107. 4. Laing PG, Ferguson AB, Hodge ES. Tissue reaction in rabbit muscle exposed to metallic implants. J Biomed Mater Res 1967;1:135149. 5. Hulbert SF, Young FA, Mathews RS, Klawitter JJ, Talbert CD, Stelling FH. Potential of ceramic materials as permanently

15.

16.

17.

18.

implantable skeletal prosthesis. J Biomed Mater Res 1970;4: 433 456. Klawitter JJ, Hulbert SF. Application of porous ceramics for the attachment of load bearing internal orthopedic application. J Biomed Mater Res Symp 1971;2:161229. Predecki P, Auslaender BA, Stephan JE. Attachment of bone to threaded implants by ingrowth and mechanical interlocking. J Biomed Mater Res 1972;6:401 412. Heimke G, Griss P, Jentschura G, Werner E. Direct anchorage of Al2O3 ceramic hip components. J Biomed Mater Res 1978; 12:57 65. Hirschhorn JS, McBeath AM, Dustoor MR. Porous titanium surgical implant materials. J Biomed Mater Res Symp 1971; 2:49 67. Linder L, Carlsson A, Marsal L, Bjursten LM, Brraanemark PI. Clinical aspects of osseointegration in joint replacement. J Bone Joint Surg 1988;70-B:550 555. Gross UM, Schmitz HJ, Strunz V. Surface activities of bioactive glass, aluminum oxide and titanium in a living environment. Ann NY Acad Sci 1988;523:211226. Hench LL, Spliner RJ, Allen WC, Greenlee TK. Bonding mechanism at the interface of ceramic prosthetic materials. J Biomed Mater Res Symp 1971;2:117141. Hench LL. Bioactive ceramics. Ann NY Acad Sci 1988;523: 54 71. Hench LL. Surface reaction kinetics and adsorption of biological moieties. In: Davies JE, editor. The bone-biomaterial interface. Toronto: Univ Toronto; 1991. p 33 48. Gross UM, Strunz V. The interface of various glasses and glass ceramics with a bony implantation bed. J Biomed Mater Res 1985;19:251271. Gross UM, MullerMai CM, Voigt C. The interface of calcium-phosphate and glass-ceramic in bone, a structural analysis. Biomater 1990;11:83 85. Kokubo T, Ito S, Sakka S, Yamamuro T. Formation of a high-strength bioactive glass-ceramic in the system MgO-CaSiO2-P2O5. J Mater Sci 1986;21:536 540. Nakamura T, Yamamuro T, Higashi S, Kokubo T, Itoo S. A new glass-ceramic for bone replacement, evaluation of its

STEM CELL TECHNOLOGY AND BIOCERAMICS

925

19. 20.

21.

22. 23.

24.

25.

26. 27. 28.

29.

30.

31.

32.

33. 34. 35.

bonding to bone tissue. J Biomed Mater Res 1985;19:685 698. Jarcho M. Calcium phosphate ceramics as hard tissue prosthetics. Clin Ortho 1981;157:259 278. Kato A, Aoki H, Tabata T, Ogiso M. Biocompatibility of apatite ceramics mandibles. Biomater Med Devices Artif Organs 1979;7:291297. Osborne JF, Newesly H. Bonding osteogenesis induced by calcium phosphate ceramic implants. In: Winter FD, Bibbons F, Plenck H, editors. Biomaterials 1980. New York: Wiley; 1982. p 5158. LeGeros RZ. Calcium phosphate materials in restorative dentistry. Adv Dent Res 1988;2:164 180. Eggli PS, Muller W, Schenk PK. Porous hydroxyapatite and tricalcium phosphate cylinders with two different pore size ranges implanted in the cancellous bone of rabbits. A comparative histomorphometric and histologic study of bony ingrowth and implant substitution. Clin Ortho 1988;232:127 138. Ohgushi H, Goldberg VM, Caplan AI. Heterotopic osteogenesis in porous ceramics induced by marrow cells. J Ortho Res 1989;7:568 578. Ohgushi H, Okumura MM, Tamai S, Shors EC, Caplan AI. Marrow cell induced osteogenesis in porous hydroxyapatite and tricalcium phosphate. J Biomed Mat Res 1990;24:1563 1570. Okumura M, Ohgushi H, Tamai S. Bonding osteogenesis in coralline hydroxyapatite combined with bone marrow cells. Biomater 1991;12:411 416. Okumura M, Ohgushi H, Tamai S, Shors EC. Primary bone formation in porous hydroxyapatite ceramic: A light and scanning elecron microscopic study. Cells Mater 1991;1:29 34. Ohgushi H, Okumura M, Yoshikawa T, Tamai S, Tabata S, Dohi Y. Regulation of bone development and the relationship to bioactivity: Osteoglastic phenotype expression of marrow stromal stem cells on the surface of bioactive materials. In: Ducheyne P, Kokubo T, van Blitterswijk CA, editors. Bonebonding biomaterials. The Netherlands: Reed Healthcare Comm; 1992. p 4756. Ohgushi H, Okumura M, Dohi Y, Tamai S, Tabata S. Materials based cell differentiation: Osteoblastic phenotype expression on bioactive materials. In: Wise DL, editor. Encyclopedia of biomaterials and bioengineering. Vol. 1. New York: Mercel Dekker; 1995. p 761771. Ohgushi H. Coral derived porous framework having different chemical compositions as a scaffold for osteoblastic differentiation. In: Liu D, Dixt V, editors. Mater sci forum porous mater tissue eng. Zurich: Trans Tech; 1997. p 209 220. LeGeros RG, Orly I, Gregoire M, Daculsi G. Substrate surface dissolution and interfacial biological mineralization. In: Davies JE, editor. The bone-biomaterial interface. Toronto: Univ Toronto; 1991. p 76 88. Ducheyne P, Radin S, Ishikawa K. The rate of calcium phosphate precipitation on metal and ceramics, and the relationship to bioactivity. In: Ducheyne P, Kokubo T, van Blitterswijk CA, editors. Bone-bonding biomaterials. The Netherlands: Reed Healthcare Comm; 1992. p 213218. Radin S, Ducheyne P. The effect of calcium phosphate composition and structure on in vitro behavior. II. Precipitation. J Biomed Mater Res 1993;27:35 44. Radin S, Ducheyne P. Effect of bioactive ceramic composition and structure on in vitro behavior. III. Porous versus dense ceramics. J Biomed Mater Res 1994;28:13031309. Kokubo T, Ito S, Huang T, Hayashi T, Sakka S, Kitugi S, Yamamura T. Ca, P-rich layer formed on high-strength bioactive glass-ceramic A-W. J Biomed Mater Res 1990;24:331 343.

36. Kokubo T, Kushitani H, Sakka S, Kitugi T, Yamamuro T. Solutions able to reproduce in vivo surface-structure changes in bioactive glass-ceramic A-W. J Biomed Mater Res 1990; 24:721734. 37. Kitugi T, Nakamura T, Yamamuro T, Kokubo T, Shibuya T, Takagi M. SEM-EPMA observation of three types of apatitecontaining glass ceramics implanted in bone. The variance of Ca-P rich layer. J Biomed Mater Res 1987;21:12551271. 38. Guilelmin G, Patat JL, Fournie J, Chetail M. The use of coral as a bone graft substitute. J Biomed Mat Res 1987;21:557 567. 39. Guillemin G, Meunier A, Dallant P, Christel P, Pouliquen JC, Sedal L. Comparison of coral resorption and bone apposition with two natural corals of different porosities. J Biomed Mat Res 1989;23:765779. 40. Fujita Y, Yamamuro T, Nakamura T, Kotani S, Ohtsuki C, Kokubo T. The bonding behavior of calcite to bone. J Biomed Mat Res 1991;25:9911003. 41. Ohgushi H, Okumura M, Yoshikawa T, Inoue K, Senpuke N, Tamai S. Bone formation process in porous calcium carbonate and hydroxyapatite. J Biomed Mat Res 1992;26:885 895. 42. Bakker D, van Blitterswijk CA, Hesseling SC, Koerten HK, Kuijpers W, Grote JJ. Biocompatibility of a polyether urethane, polypropylene oxide and a polyether polyester copolymer. A qualitative and quantitative study of three aaloplastic typanic membrane materials in the rat middle ear. J Biomed Mater Res 1990;24:489 515. 43. van Blitterswijk CA, Hesseling SC, van den Brink J, Leenders H, Bakker D. Polymer reactions resulting in bone bonding; A review of the biocompatibility of Polyactive. In: Davies JE, editor. The bone-biomaterial interface. Toronto: Univ Toronto; 1991. p 295307. 44. Radder AM, van Blitterswijk CA, Leenders H, Inoue K, Okumura M, Ohgushi H. Gene expression and protein activity in bone-bonding and non-bonding PEO/PBT copolymers. J Mater Sci 1994;5:582586. 45. Tretinnikov ON, Kato K, Ikada Y. In vitro hydroxyapatite deposition onto a lm surface-grated with organophosphate polymer. J Biomed Mater Res 1994;28:13651373. 46. Bruder SP, Caplan AI. First bone formation and the dissection of an osteogenic lineage in the embryonic chick tibia is revealed by monoclonal antibodies against osteoblasts. Bone 1989;10:359 375. 47. Bruder SP, Caplan AI. Terminal differentiation of osteogenic cells in the embryonic chick tibia is revealed by a monoclonal antibody against osteocytes. Bone 1990;11:189 198. 48. Owen M. Lineage of osteogenic cells and their relationship to the stromal system. In: Peck WA, editor. Bone and mineral research. London: Elsevier Science BV; 1985;3:125. 49. Friedenstein AJ, Petrakova KV, Kurolesova AI, Frolova GD. Heterotopic transplants of bone marrow. Analysis of precursor cells for osteogenic and haemopoietic tissues. Transplant 1968;6:230 247. 50. Caplan AI. Mesenchymal stem cells. J Ortho Res 1991;9:641 650. 51. Haynesworth SE, Baber MA, Caplan AI. Cell surface antigens on human marrow-derived mesenchymal cells are detected by monoclonal antibodies. Bone 1992;13:69 80. 52. Nakahara H, Dennis JE, Bruder SP, Haynesworth SE, Lennon DP, Caplan AI. In vitro differentiation of bone and hypertrophic cartilage from periosteal-derived cells. Exp Cell Res 1991;195:492503. 53. Caplan AI, Fink DJ, Goto T, Linton AE, Young RG, Wakitani S, Goldberg VM, Haynesworth SE. Mesenchymal stem cells and tissue repair. In: Jackson D, Arnoczky S, Woo S, Frank C, editors. The anterior cruciate ligament: current and future concepts. New York: Raven; 1993. p 405 417.

926

OHGUSHI AND CAPLAN

54. Caplan AI. Mesenchymal stem cell-mediated cartilage and bone repair. In: Davidovitch Z, editor. The biological mechanisms of tooth movement and craniofacial adaptation. EBSCO Pub; 1992. p 433 438. 55. Okumura M, Ohgushi H, Takakura Y, van Blitterswijk CA, Koerten K. Analysis of primary bone formation in porous alumina: A uorescence and scanning electron microscopic study of marrow cells induced osteogenesis. Biomed Mat Eng 1992;2:191201. 56. Okumura M, Ohgushi H, Ducheyne P. Porous titanium felt as a carrier of osteogenic cells. In: Ducheyne P, Christiansen D, editors. Bioceramics. Vol. 6. Oxford: ButterworthHeinemann; 1993. p 395310. 57. Takaoka T, Okumura M, Ohgushi H, Inoue K, Takakura Y, Tamai S. Histological and biochemical evaluation of osteogenic response in porous hydroxyapatite coated alumina ceramics. Biomater 1996;17:1499 1505. 58. Yoshikawa T, Ohgushi H, Okumura M, Tamai S, Dohi Y, Moriyama T. Biochemical and histological sequences of membranous ossication in ectopic site. Calcif Tissue Int 1992;50: 184 188. 59. Ohgushi H, Dohi Y, Tamai S, Tabata S. Osteogenic differentiation of marrow stromal stem cells in porous hydroxyapatite ceramics. J Biomed Mat Res 1993;27:14011407. 60. Okumura M, Ohgushi H, Dohi T, Katsuda T, Tamai S, Koerten HK, Tabata S. Osteoblastic phenotype expression on the surface of hydroxyapatite ceramics. J Biomed Mat Res 1997;37: 122129. 61. Ducheyne P, Bianco P, Radin S, Schepers E. Bioactive materials, mechanisms and bioengineering considerations. In: Ducheyne P, Kokubo T, van Blitterswijk CB, editors. Bonebonding biomaterials. The Netherlands: Reed Healthcare Comm; 1992. p 112. 62. Dennis JE, Haynesworth SE, Young RG, Caplan AI. Osteogenesis in marrow-derived mesenchymal cell porous ceramic composites transplanted subcutaneously: effect of bronectin and laminin on cell retention and rate of osteogenic expression. Cell Transpl 1992;1:2332. 63. Dennis JE, Caplan AI. Porous ceramic vehicles for rat-marrow-derived (Rattus norvegicus) osteogenic cell delivery, effects of pre-treatment with bronectin or laminin. J Oral Implant 1993;19:106 115. 64. Dennis JE, Konstantakos EK, Arm D, Caplan AI. In vivo osteogenesis assay: a rapid method for quantitative analysis. Biomater 1998;19:13231328. 65. Nakahara H, Goldberg VM, Caplan AI. Culture-expanded human periosteal-derived cells exhibit osteochondral potential in vivo. J Ortho Res 1991;9:465 476. 66. Ohgushi H, Okumura MM. Osteogenic ability of rat and human marrow cells in porous ceramics. Acta Ortho Scand 1990;61:431 434. 67. Yoshikawa T, Suwa Y, Ohgushi H, Tamai S, Ichijima K. Self setting hydroxyaptite cement as a carrier for bone-forming cells. Biomed Mat Eng 1996;6:345351. 68. Yoshikawa T, Ohgushi H, Tamai S. Immediate bone forming capability of prefabricated osteogenic hydroxyapatite. J Biomed Mat Res 1996;32:481 492. 69. Goshima J, Goldberg VM, Caplan AI. The osteogenic potential of culture-expanded rat marrow mesenchymal cells assayed in vivo in calcium phosphate ceramic blocks. Clin Ortho 1991;262:298 311. 70. Haynesworth SE, Goshima J, Goldberg VM, Caplan AI. Characterization of cells with osteogenic potential from human marrow. Bone 1992;13:81 88. 71. Lennon DP, Haynesworth SE, Young RG, Dennis JE, Caplan AI. A chemically dened medium supports proliferation and maintains osteochondrogenic potential of rat marrow-derived mesenchymal stem cells. Exp Cell Res 1995;219:211222.

72. Cassiede P, Dennis JE, Ma F, Caplan AI. Osteochondrogenic potential of marrow mesenchymal progenitor cells exposed to TGF-1 or PDGF-BB as assayed in vivo and in vitro. J Bone Min Res 1996;11:1264 1273. 73. Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP. Osteogenic differentiation of puried, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 1997;64:295 312. 74. Lennon DP, Haynesworth SE, Bruder SP, Jaiswall N, Caplan AI. Human and animal mesenchymal progenitor cells from bone marrow: Identication of serum for optimal selection and proliferation. In Vitro Cell Develop Biol 1996;32:602 611. 75. Johnstone B, Hering TM, Goldberg VM, Yoo JU, Caplan AI. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 1998;238:265272. 76. Goshima J, Goldberg VM, Caplan AI. The origin of bone formed in composite grafts of porous calcium phosphate ceramic loaded with marrow cells. Clin Ortho Rel Res 1991;269: 274 283. 77. Ohgushi H, Dohi Y, Yoshikawa T, Tamai S, Tabata S, Okunaga K, Shibuya T. Osteogenic differentiation of cultured marrow stromal stem cells on the surface of bioactive glass ceramics. J Biomed Mat Res 1996;32:341348. 78. Ohgushi H, Dohi Y, Kaduda T, Tamai S, Tabata S, Suwa Y. In vitro bone formation by rat marrow cell culture. J Biomed Mat Res 1996;32:333340. 79. Yoshikawa T, Ohgushi H, Dohi Y, Davies JE. Viable bone formation in porous hydroxyapatite: marrow-derived in vitro bone on the surface of ceramic. Biomed Mat Eng 1997;7:49 58. 80. Kadiyala S, Jaiswal N, Bruder SP. Culture-expanded, bone marrow-derived mesenchymal stem cells can regenerate a critical-sized segmental bone defect. Tissue Eng 1997;3:173185. 81. Bruder SP, Kraus KH, Goldberg VM, Kadiyala S. The effect of autologous mesenchymal stem cell implants in the healing of canine segmental bone defects. J Bone Joint Surg, to appear. 82. Ohgushi H, Dohi Y, Okumura M, Inoue KK, Tamai S, Tabata S. Molecular biology as a tool to investigate cell/material interaction. In: Ducheyne P, Christiansen D, editors. Bioceramics. Vol. 6. Oxford: ButterworthHeinemann; 1993. p 27 31. 83. Sodek J, Zang Q, Goldberg HA, Domenicucci C, Kasugai S, Wrana JL, Shapiro H, Chen J. Non-collagenous bone proteins and their role in substrate-induced bioactivity. In: Davies JE, editor. The bone-biomaterial interface. Toronto: Univ Toronto; 1991. p 97108. 84. Yabe T, Nemoto A, Uemura T. Recognition of osteopontin by rat bone marrow derived osteoblastic primary cells. Biosci Biotech Biochem 1997;61:754 756. 85. Seitz TL, Noonan KD, Hench LL, Noonan NE. Effect of bronectin on the adhesion of an established cell line to a surface reactive biomaterial. J Biomed Mat Res 1982;16:195 207. 86. Okumura A, Ohgushi H, Inoue KK, Tamai S, Dohi S, Shibuya T, Komatsudani S. Osteogenic response of bone marrow cells in porous A-W glass ceramic. In: Andersson OH, Happonen RP, YliUrpo A, editors. Bioceramics. Vol. 7. Oxford: ButterworthHeinemann; 1994. p 337340. 87. Hanawa T. Titanium and its oxide lm: A substrate for formation of apatite. In: Davies JE, editor. The bone-biomaterial interface. Toronto: Univ Toronto; 1991. p 49 61. 88. Kim HM, Miyaji F, Kokubo T, Nakamura T. Bonding strength of bonelike apatite layer to Ti metal substrate. J Biomed Mater Res 1997;38:121127. 89. Yan WQ, Nakamura T, Kobayashi M, Kim HM, Miyaji F, Kokubo T. Bonding of chemically treated titanium implants to bone. J Biomed Mater Res 1997;37:267275.

STEM CELL TECHNOLOGY AND BIOCERAMICS

927

90. Maniatopoulos C, Sodek J, Melcher AH. Bone formation in vitro by stromal cells obtained from marrow of young adult rats. Cell Tissue Res 1988;254:317330. 91. Davies JE, Chernecky R, Lowenberg B, Shiga A. Deposition and resorption of calcied matrix in vitro by rat marrow cells. Cells Mater 1991;1:315. 92. de Bruijin JD, van den Brink J, Bovell YP. Development of human bone marrow culture to examine the interface between apatite and bone formed in vitro. In: Kokubo T, Nakamura T, Miyaji F, editors. Bioceramics. Vol. 9. Amsterdam: Elsevier Science; 1996. p 45 48. 93. Hanada K, Dennis JE, Caplan AI. Stimulatory effects of basic broblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2) on osteogenic differentiation of rat bone marrow-derived, mesenchymal stem cells (MSCs). J Bone Min Res 1997;12:1606 1614. 94. Yoshikawa T, Ohgushi H, Akahane M, Tamai S, Ichijima K. Analysis of gene expression in osteogenic cultured marrow/ hydroxyapatite construct implanted at ectopic sites: a comparison with osteogenic ability of cancellous bone. J Biomed Mat Res 1998;41:568 573. 95. Ohgushi H, Goldberg VM, Caplan AI. Repair of bone defects with marrow and porous ceramic. (Experiments in rats). Acta Ortho Scand 1989;60:334 339. 96. Ohgushi H, Okumura M, Miyauchi Y, Mii Y, Tamai S. Composite grafts of human bone marrow cells and porous ceramics. In: Hulbert JE, Hulbert SF, editors. Bioceramics. Vol. 3. Rose Hulman Inst Tech; 1992. p 279 286. 97. Wakitani S, Goto T, Pineda SJ, Young RG, Mansour JM, Goldberg VM, Caplan AI. Mesenchymal cell-based repair of large full-thickness defects of articular cartilage and underlying bone. J Bone Joint Surg 1994;76:579 592. 98. Caplan AI, Elyaderani M, Mochizuki Y, Wakitani S, Goldberg VM. The principles of cartilage repair/regeneration. Clin Ortho Rel Res 1997;342:254 269. 99. Haynesworth SE, Goldberg VM, Caplan AI. Diminution of the number of mesenchymal stem cells as a cause for skeletal aging. In: Buckwalter JA, Goldberg VM, Woo SLY, editors. Musculoskeletal soft-tissue aging: Impact on mobility. Am Acad Ortho Surg; 1994. p 79 87. 100. Tabuchi C, Simmons DJ, Fausto A, Russell JE, Binderman I, Avioli LV. Bone decit in ovariectomized rats: functional contribution of the marrow stromal cell population and the effect of oral dihydrotachysterol treatment. J Clin Invest 1986; 78:637 642. 101. Inoue K, Ohgushi H, Yoshikawa T, Okumura M, Sempuku T, Tamai S, Dohi Y. The effect of aging on bone formation in porous hydroxyapatite. J Bone Min Res 1997;12:989 994. 102. Nakahara H, Goldberg VM, Caplan AI. Culture-expanded human periosteal-derived cells exhibit osteochondral potential in vivo. J Ortho Res 1991;9:465 476. 103. Young RG, Butler DL, Weber W, Gordon SL, Fink DJ, Caplan AI. The use of mesenchymal stem cells in a collagen matrix for achilles tendon repair. J Ortho Res 1998;16:406 413. 104. Hasegawa Y, Ohgushi H, Ishimura M, Habata T, Tamai S, Tomita N, Ikada Y. Marrow cell culture on poly-L-lactic acid fabrics. Clin Ortho 1999;358:235243.

105. Pilliar RM. Powder metal-made orthopedic implants with porous surface for xation by tissue ingrowth. Clin Ortho 1983; 176:4251. 106. Haddad RJ, Cook SD, Thomas KA. Biological xation of porous-coated implants. J Bone Joint Surg 1987;69-A:1459 1466. 107. Knowles JC, Gross K, Berndt CC, Boneld W. Structural changes of thermally sprayed hydroxyapatite investigated by retrieved analysis. Biomater 1996;17:639 645. 108. Overgaard S, Lind M, Glerup H, Grundvig S, Bunger C, Soballe K. Hydroxyapatite and uorapatite coatings for xation of weight loaded implants. Clin Ortho 1997;336: 286 296. 109. Dean JC, Tisdel CL, Goldberg VM, Parr J, Davy D, Stevenson S. Effects of hydroxyapatite tricalcium phosphate coating and intracancellous placement on bone ingrowth in titanium bermetal implants. J Arthroplasty 1995;10:830 838. 110. Geesink RG, Hoefnagels NH. Six-year results of hydroxyapatite-coated total hip replacement. J Bone Joint Surg 1995;77B:534 547. 111. Oonishi H, Yamamoto M, Ishimaru H, Tsuji E, Kushitani S, Aono M, Ukon Y. The effect of hydroxyapatite coating on bone growth into porous titanium alloy implants. J Bone Joint Surg 1989;71-B:213216. 112. Muschler GF, Boehm C, Easley K. Aspiration to obtain osteoblast progenitor cells from human bone marrow: the inuence of aspiration volume. J Bone Joint Surg 1997;79:1699 1710. 113. Yoshikawa T, Ohgushi H, Nakajima H, Akahane M, Tamai S, Ichijima K. A long term implantation of cultured bone in porous hydroxyapatite. In: Sedel L, Ray C, editors. Bioceramics. Vol. 10. London: Elsevier Science; 1997. p 117120. 114. Ohgushi H, Dohi Y, Tabata S, Okumura MM, Tamai S. Persistence of osteoblast phenotype expression in marrow/ hydroxyapatite composite. In: Yamamuro T, Kokubo T, Nakamura T, editors. Bioceramics. Vol. 5. Kyoto Japan: Kobunshi Kankokai; 1992. p 119 124. 115. Allay JA, Dennis JE, Haynesworth SE, Majumdar M, Clapp DW, Caplan AI, Gerson SL. LacZ and IL-3 expression in vivo after retroviral transduction of marrow-derived human osteogenic mesenchymal progenitors. Human Gene Ther 1997;8: 14171427. 116. Caplan AI. Osteogenesis imperfecta, rehabilitation medicine and fundamental research. Conn Tissue Res 1995;31:S9-S14. 117. Caplan AI, Fink DJ, Bruder SP, Young G, Butler DL. The regeneration of skeletal tissues using mesenchymal stem cells. In: Patrick CW Jr, Mikos AG, McIntire LV, editors. Frontiers in tissue engineering. New York: Elsevier Science; 1998. p 471 480. 118. Haynesworth SE, Reuben D, Caplan AI. Cell-based tissue engineering therapies, the inuence of whole body physiology. Adv Drug Deliv Rev 1998;33:314. 119. Caplan AI, Bruder SP. Cell and molecular engineering of bone regeneration. In: Lanza RP, Chick WL, Langer R, editors. Principles of tissue engineering. Springer, NY: RG Landes; 1996. p 599 618.