Renny T. Franceschi

Isolation and Characterization of MC3T3-E1 Preosteoblast Subclones with Distinct In Vitro and In Vivo Differentiation/Mineralization Potential†

A series of subclonal cell lines with high or low differentiation/mineralization potential after growth in the presence of ascorbic acid (AA) were derived from murine MC3T3‐E1 cells. Subclones were characterized in terms of their ability to mineralize a collagenous extracellular matrix both in vitro and in vivo and express osteoblast‐related genes. When compared with nonmineralizing cells, mineralizing subclones selectively expressed mRNAs for the osteoblast markers, bone sialoprotein (BSP), osteocalcin (OCN), and the parathyroid hormone (PTH)/parathyroid hormone‐related protein (PTHrP) receptor. In contrast, alkaline phosphatase mRNA was present in certain nonmineralizing as well as mineralizing subclones, suggesting that its expression may be subject to different controls from other osteoblast markers. Only highly differentiating subclones exhibited strong AA‐dependent induction of a transiently transfected OCN promoter‐luciferase reporter gene, indicating that there was a good correlation between mRNA levels and transcriptional activity. Consistent with its postulated role in biomineralization, BSP as measured by Western blotting was only present in mineralizing subclones. After implantation into immunodeficient mice, highly differentiating subclones formed bone‐like ossicles resembling woven bone, while poorly differentiating cells only produced fibrous tissue. Interestingly, subclones with both high and low differentiation potential produced similar amounts of collagen in culture and expressed comparable basal levels of mRNA encoding Osf2/Cbfa1, an osteoblast‐related transcription factor. Although some strongly differentiating cells exhibited a modest AA‐dependent up‐regulation of Osf2/Cbfa1 mRNA, there was no clear relationship between levels of this message and induction of mRNAs for other differentiation markers. Thus, the mere presence of Osf2/Cbfa1 in a subclone was not sufficient for osteoblast differentiation. These subclones will be very useful for studying critical events in osteoblast differentiation and mineralization.

Regulation of the osteoblast-specific transcription factor, Runx2: Responsiveness to multiple signal transduction pathways

The Cbfa1/Runx2 is an important transcription factor necessary for osteoblast differentiation and bone formation. However, the signaling pathways regulating Runx2 activity are just beginning to be understood. Inconsistencies between Runx2 mRNA or protein levels and its transcriptional activity suggests that posttranslational modification and/or protein‐protein interactions may regulate this factor. Runx2 can be phosphorylated and activated by the mitogen‐activated protein kinase (MAPK) pathway. This pathway can be stimulated by a variety of signals including those initiated by extracellular matrix (ECM), osteogenic growth factors like bone morphogenic proteins (BMPs) and fibroblast growth factor‐2 (FGF‐2), mechanical loading and hormones such as parathyroid hormone (PTH). Protein kinase A (PKA) may also phosphorylate/activate Runx2 under certain conditions. In addition, Runx2 activity is enhanced by protein‐protein interactions as are seen with PTH‐induced Runx2/AP‐1 and BMP‐mediated Runx2/Smads interactions. Mechanisms for interaction with Runx2 are complex including binding of distinct components such as AP‐1 factors and Smads proteins to separate DNA regions in target gene promoters and direct physical interactions between Runx2 and AP‐1/Smad factors. Post‐translational modifications such as phosphorylation may influence interactions between Runx2 and other nuclear factors. These findings suggest that Runx2 plays a central role in coordinating multiple signals involved in osteoblast differentiation.

Bone morphogenetic proteins, extracellular matrix, and mitogen-activated protein kinase signaling pathways are required for osteoblast-specific gene expression and differentiation in MC3T3-E1 cells

Osteoblasts secrete a complex extracellular matrix (ECM) containing collagenous and noncollagenous proteins, bone morphogenetic proteins (BMPs), and growth factors. Osteoblast‐specific gene expression requires ascorbic acid (AA)‐dependent assembly of a collagenous ECM. Matrix responsiveness requires an α2β1 integrin‐collagen interaction and mitogen‐activated protein kinase (MAPK) activity, which phosphorylates and activates the osteoblast‐specific transcription factor Cbfa1. This study examines interactions between this integrin/MAPK‐mediated pathway and signals initiated by BMPs contained in the osteoblast matrix. MC3T3‐E1 cells were shown to constitutively express BMP‐2, BMP‐4, and BMP‐7. Noggin, a specific BMP inhibitor, reversibly blocked AA‐induced gene expression, indicating that BMP production by MC3T3‐E1 cells was necessary for differentiation. The ability of exogenously added BMP‐2, BMP‐4, or BMP‐7 to stimulate osteocalcin (OCN) and bone sialoprotein (BSP) mRNAs or OCN promoter activity was synergistically increased in cells that were actively synthesizing an ECM (i.e., were grown in the presence of AA). A minimum of 4 days of ECM accumulation was required for this synergistic response to be observed. Neither BMP‐7, AA, nor a combination of these two treatments had major effects on Cbfa1 messenger RNA (mRNA) or protein levels, as would be expected if regulation was mainly at the posttranscriptional level. U0126, a specific inhibitor of MAPK/extracellular signal‐regulated kinase (MEK), blocked AA‐ or BMP‐7/AA‐dependent gene expression in a time‐ and dose‐dependent manner that was closely correlated with inhibition of extracellular signal‐regulated kinase (ERK) phosphorylation. This work establishes that autocrine BMP production as well as integrin‐mediated cell‐collagen interactions are both required for osteoblast differentiation, and both these pathways require MAP kinase activity.

Engineering new bone tissue in vitro on highly porous poly(α-hydroxyl acids)/hydroxyapatite composite scaffolds

Engineering new bone tissue with cells and a synthetic extracellular matrix (scaffolding) represents a new approach for the regeneration of mineralized tissues compared with the transplantation of bone (autografts or allografts). In the present work, highly porous poly(L-lactic acid) (PLLA) and PLLA/hydroxyapatite (HAP) composite scaffolds were prepared with a thermally induced phase separation technique. The scaffolds were seeded with osteoblastic cells and cultured in vitro. In the pure PLLA scaffolds, the osteoblasts attached primarily on the outer surface of the polymer. In contrast, the osteoblasts penetrated deep into the PLLA/HAP scaffolds and were uniformly distributed. The osteoblast survival percentage in the PLLA/HAP scaffolds was superior to that in the PLLA scaffolds. The osteoblasts proliferated in both types of the scaffolds, but the cell number was always higher in the PLLA/HAP composite scaffolds during 6 weeks of in vitro cultivation. Bone-specific markers (mRNAs encoding bone sialoprotein and osteocalcin) were expressed more abundantly in the PLLA/HAP composite scaffolds than in the PLLA scaffolds. The new tissue increased continuously in the PLLA/HAP composite scaffolds, whereas new tissue formed only near the surface of pure PLLA scaffolds. These results demonstrate that HAP imparts osteoconductivity and the highly porous PLLA/HAP composite scaffolds are superior to pure PLLA scaffolds for bone tissue engineering.

Inhibition of osteoblastic bone formation by nuclear factor-κB

An imbalance in bone formation relative to bone resorption results in the net bone loss that occurs in osteoporosis and inflammatory bone diseases. Although it is well known how bone resorption is stimulated, the molecular mechanisms that mediate impaired bone formation are poorly understood. Here we show that the time- and stage-specific inhibition of endogenous inhibitor of κB kinase (IKK)–nuclear factor-κB (NF-κB) in differentiated osteoblasts substantially increases trabecular bone mass and bone mineral density without affecting osteoclast activities in young mice. Moreover, inhibition of IKK–NF-κB in differentiated osteoblasts maintains bone formation, thereby preventing osteoporotic bone loss induced by ovariectomy in adult mice. Inhibition of IKK–NF-κB enhances the expression of Fos-related antigen-1 (Fra-1), an essential transcription factor involved in bone matrix formation in vitro and in vivo. Taken together, our results suggest that targeting IKK–NF-κB may help to promote bone formation in the treatment of osteoporosis and other bone diseases.An imbalance in bone formation relative to bone resorption results in the net bone loss in osteoporosis and inflammatory bone diseases. While it is well known how bone resorption is stimulated, the molecular mechanisms that mediate impaired bone formation are poorly understood. Here we show that the time- and stage-specific inhibition of endogenous IκB kinase (IKK)/nuclear factor-kappa B (NF-κB) NF-κB in differentiated osteoblasts significantly increases trabecular bone mass and bone mineral density without affecting osteoclast activities in young mice. Moreover, the inhibition of IKK/NF-κB in differentiated osteoblasts maintains bone formation, thereby preventing osteoporotic bone loss induced by ovariectomy (OVX) in adult mice. The inhibition of IKK/NF-κB enhances the expression of Fra-1, an essential factor for bone matrix formation in vitro and in vivo. Taken together, our results suggest that targeting IKK/NF-κB may help to promote bone formation in the treatment of osteoporosis and other bone diseases.

Gene Therapy-Directed Osteogenesis: BMP-7-Transduced Human Fibroblasts Form Bone in Vivo

An ex vivo gene therapy strategy was used to achieve localized skeletal regeneration in vivo. When an adenovirus vector engineered to express bone morphogenetic protein 7 transduced human gingival fibroblasts or rat dermal fibroblasts, these nonosteogenic tissues formed bone and supported the development of hematopoietic tissue when transplanted into immunocompromised mice. Transduced gingival fibroblasts formed marrow-containing ossicles in 100% of transplants after 1-2 weeks in vivo (n = 30). Immunostaining with murine and human-specific antisera raised against osteonectin and in situ hybridization of human-specific Alu genomic sequence demonstrated that the newly formed bone organ was a chimera of both the human donor and the mouse recipient cells. In experiments of greater clinical relevance, AdCMVBMP-7-transduced dermal fibroblasts repaired critical size skeletal defects in rat calvariae (n = 6). The results of this study suggest a bifunctional role of BMP-7-transduced fibroblasts. The transduced, nonosteogenic cells not only secreted biologically active BMP-7 in vitro and in vivo, but also differentiated into bone-forming cells in vivo. This model exploits the use of an easily biopsied, self-regenerating tissue such as gingiva or skin and suggests that local regeneration of tissues by ex vivo gene therapy may not require that autogenous cells be cultured from the tissue that is to be regenerated.

Gene therapy for bone formation: In vitro and in vivo osteogenic activity of an adenovirus expressing BMP7

Bone morphogenetic proteins (BMPs) are well‐established agents for inducing orthotopic and ectopic bone formation. However, their clinical usefulness as regenerative agents may be limited by a short in vivo half‐life and low specific activity. BMP gene therapy is an alternative route for exploiting the bone‐inductive activity of this class of molecules. To test the feasibility of this approach, we examined the osteogenic activity of AdCMV‐BMP7, an adenovirus containing BMP7 cDNA under control of the CMV promoter that was constructed using Cre/lox recombination (Hardy et al. [1997] J. Virol. 71:1842–1849). Adenovirus vectors were shown to readily infect a wide variety of cell types in vitro including osteoblasts, fibroblasts, and myoblasts. COS7 cells transduced with AdCMV‐BMP7 produced high levels of BMP‐7 (approximately 0.5 μg/106 cells). Furthermore, transduction of C2C12 murine myoblast cells with AdCMVBMP‐7 suppressed the muscle phenotype and induced in vitro osteoblast differentiation. To test its in vivo biological activity, AdCMV‐BMP7 was mixed with a bovine bone‐derived collagen carrier (108 plaque‐forming units virus/site) and was implanted into mouse muscle and dermal pouches. In both cases, an ossicle containing cortical and trabecular bone and a clearly defined marrow cavity formed at the site of virus implantation within 4 weeks. These data demonstrate that AdCMV‐BMP7 transduced cells produce biologically active BMP‐7 both in vitro and in vivo and show that gene therapy by direct viral transduction using a virus/matrix implant may be a viable route for stimulating bone regeneration. J. Cell. Biochem. 78:476–486, 2000.

BMP signaling is required for RUNX2-dependent induction of the osteoblast phenotype

RUNX2 expression in mesenchymal cells induces osteoblast differentiation and bone formation. BMP blocking agents were used to show that RUNX2‐dependent osteoblast differentiation and transactivation activity both require BMP signaling and, further, that RUNX2 enhances the responsiveness of cells to BMPs.

Engineered Bone Development from a Pre-Osteoblast Cell Line on Three-Dimensional Scaffolds

Bone regeneration is based on the hypothesis that healthy progenitor cells, either recruited or delivered to an injured site, can ultimately regenerate lost or damaged tissue. Three-dimensional porous polymer scaffolds may enhance bone regeneration by creating and maintaining a space that facilitates progenitor cell migration, proliferation, and differentiation. As an initial step to test this possibility, osteogenic cells were cultured on scaffolds fabricated from biodegradable polymers, and bone development on these scaffolds was evaluated. Porous polymer scaffolds were fabricated from biodegradable polymers of lactide and glycolide. MC3T3-E1 cells were statically seeded onto the polymer scaffolds and cultured in vitro in the presence of ascorbic acid and beta-glycerol phosphate. The cells proliferated during the first 4 weeks in culture and formed a space-filling tissue. Collagen messenger RNA levels remained high in these cells throughout the time in culture, which is consistent with an observed increase in collagen deposition on the polymer scaffold. Mineralization of the deposited collagen was initially observed at 4 weeks and subsequently increased. The onset of mineralization corresponded to increased mRNA levels for two osteoblast-specific genes: osteocalcin and bone sialoprotein. Culture of cell/polymer constructs for 12 weeks led to formation of a three-dimensional tissue with architecture similar to that of native bone. These studies demonstrate that osteoblasts within a three-dimensional engineered tissue follow the classic differentiation pathway described for two-dimensional culture. Polymer scaffolds such as these may ultimately be used clinically to enhance bone regeneration by delivering or recruiting progenitor cells to the wound site.

In vitro and in vivo synergistic interactions between the Runx2/Cbfa1 transcription factor and bone morphogenetic protein-2 in stimulating osteoblast differentiation.

Bone regeneration requires interactions between a number of factors including bone morphogenetic proteins (BMPs), growth factors, and transcriptional regulators such as Runx2/Cbfa1 (Runx2). Because each component may provide a unique contribution to the overall osteogenic response, we hypothesized that bone formation may be enhanced by using combinations of complimentary factors. As an initial test of this concept, interactions between BMP2 and Runx2 were examined using adenovirus‐based expression vectors (AdCMV‐Runx2, AdCMV‐BMP2) in the pluripotent C3H10T1/2 cell line. Cells transduced with AdCMV‐Runx2 strongly expressed osteoblast markers, such as alkaline phosphatase and osteocalcin, but formed only a weakly mineralized extracellular matrix in vitro, whereas cells transduced with AdCMV‐BMP2 exhibited higher levels of mineralization, but only expressed low levels of Runx2 and osteocalcin mRNA. Significantly, when cells were transduced with optimal titers of both viruses, osteoblast differentiation was stimulated to levels that were 10‐fold greater than those seen with either AdCMV‐Runx2 or AdCMV‐BMP2 alone. To measure in vivo osteogenic activity, virally transduced cells were subcutaneously implanted into immunodeficient mice. Cells transduced with control virus produced only fibrous tissue while those with AdCMV‐Runx2 produced limited amounts of both cartilage and bone. In contrast, cells transduced with either AdCMV‐BMP2 alone or AdCMV‐BMP2 plus AdCMV‐Cbfa1 generated large ossicles containing cartilage, bone, and a marrow cavity. However, ossification in the AdCMV‐BMP2 plus AdCMV‐Cbfa1 group was more extensive in that both mineral content and fractional bone area were greater than that seen in the AdCMV‐BMP2 group. Thus, the increased osteoblast differentiation observed with combined adenovirus treatment in vitro is also manifested by increased bone formation in vivo. These results suggest that Runx2 and BMP2 have distinct, but complementary, roles in osteogenesis and that their combined actions may be necessary for optimal bone formation.