Vicki Rosen

BONE MORPHOGENETIC PROTEIN AND BONE MORPHOGENETIC PROTEIN GENE FAMILY IN BONE FORMATION AND REPAIR

The bone morphogenetic proteins are secreted signalling molecules that belong to the transforming growth factor beta family of growth and differentiation factors. Individual bone morphogenetic proteins are prominent at many sites during embryogenesis and are likely to be key regulators of early development and organogenesis. In vertebrates, one of the functions of bone morphogenetic like proteins is to induce formation of bone, cartilage, and connective tissues associated with the skeleton. This osteoinductive ability has led to the use of bone morphogenetic proteins as therapeutic agents for creation of new bone useful in treatment of skeletal injuries and diseases, and in oral and maxillofacial applications.

BMP2 activity, although dispensable for bone formation, is required for the initiation of fracture healing

Adult bones have a notable regenerative capacity. Over 40 years ago, an intrinsic activity capable of initiating this reparative response was found to reside within bone itself, and the term bone morphogenetic protein (BMP) was coined to describe the molecules responsible for it. A family of BMP proteins was subsequently identified, but no individual BMP has been shown to be the initiator of the endogenous bone repair response. Here we demonstrate that BMP2 is a necessary component of the signaling cascade that governs fracture repair. Mice lacking the ability to produce BMP2 in their limb bones have spontaneous fractures that do not resolve with time. In fact, in bones lacking BMP2, the earliest steps of fracture healing seem to be blocked. Although other osteogenic stimuli are still present in the limb skeleton of BMP2-deficient mice, they cannot compensate for the absence of BMP2. Collectively, our results identify BMP2 as an endogenous mediator necessary for fracture repair.

The non-osteogenic mouse pluripotent cell line, C3H10T1/2, is induced to differentiate into osteoblastic cells by recombinant human bone morphogenetic protein-2.

The possibility that the non-osteogenic mouse pluripotent cell line, C3H10T1/2 (10T1/2), could be induced to differentiate into osteogenic cells by various hormones and cytokines was examined in vitro. Of a number of agents tested, recombinant human bone morphogenetic protein-2 (rhBMP-2) and retinoic acid induced alkaline phosphatase (ALP) activity in 10T1/2 cells. rhBMP-2 also induced mRNA expression of ALP in the cells. Dexamethasone, 1 alpha, 25-dihydroxyvitamin D3, transforming growth factor-beta 1 and insulin-like growth factor-I did not stimulate ALP activity. Treatment with rhBMP-2 greatly induced cAMP production in response to parathyroid hormone in 10T1/2 cells. No ALP activity was induced in NIH3T3 fibroblasts treated with rhBMP-2 or retinoic acid. These results indicate that 10T1/2 cells have a potential to differentiate into osteogenic cells under the control of BMP-2.

Ectopic induction of tendon and ligament in rats by growth and differentiation factors 5, 6, and 7, members of the TGF-beta gene family.

Little is known about the regulatory signals involved in tendon and ligament formation, and this lack of understanding has hindered attempts to develop biologically based therapies for tendon and ligament repair. Here we report that growth and differentiation factors (GDFs) 5, 6, and 7, members of the TGF-beta gene superfamily that are most related to the bone morphogenetic proteins, induce neotendon/ligament formation when implanted at ectopic sites in vivo. Analysis of tissue induced by GDF-5, 6, or 7, containing implants by currently available morphological and molecular criteria used to characterize tendon and ligament, adds further evidence to the idea that these GDFs act as signaling molecules during embryonic tendon/ligament formation. In addition, comparative in situ localizations of the GDF-5, 6, and 7 mRNAs suggest that these molecules are important regulatory components of synovial joint morphogenesis.

Genetic Analysis of the Roles of BMP2, BMP4, and BMP7 in Limb Patterning and Skeletogenesis

Bone morphogenetic protein (BMP) family members, including BMP2, BMP4, and BMP7, are expressed throughout limb development. BMPs have been implicated in early limb patterning as well as in the process of skeletogenesis. However, due to complications associated with early embryonic lethality, particularly for Bmp2 and Bmp4, and with functional redundancy among BMP molecules, it has been difficult to decipher the specific roles of these BMP molecules during different stages of limb development. To circumvent these issues, we have constructed a series of mouse strains lacking one or more of these BMPs, using conditional alleles in the case of Bmp2 and Bmp4 to remove them specifically from the limb bud mesenchyme. Contrary to earlier suggestions, our results indicate that BMPs neither act as secondary signals downstream of Sonic Hedghog (SHH) in patterning the anteroposterior axis nor as signals from the interdigital mesenchyme in specifying digit identity. We do find that a threshold level of BMP signaling is required for the onset of chondrogenesis, and hence some chondrogenic condensations fail to form in limbs deficient in both BMP2 and BMP4. However, in the condensations that do form, subsequent chondrogenic differentiation proceeds normally even in the absence of BMP2 and BMP7 or BMP2 and BMP4. In contrast, we find that the loss of both BMP2 and BMP4 results in a severe impairment of osteogenesis.

Heterodimeric bone morphogenetic proteins show enhanced activity in vitro and in vivo.

The bone morphogenetic proteins (BMPs), a subgroup of the TGF-beta gene super-family, are dimeric molecules involved in the growth, differentiation and repair of a wide variety of tissues. Based on the observation that several of the BMPs co-purify when isolated from bovine bone and that a pattern of co-localization exists during mouse embryogenesis, we co-expressed various combinations of BMPs in Chinese hamster ovary cells to test for possible heterodimer formation and activity. Transient co-expression of BMP-2 with either BMP-5, BMP-6 or BMP-7, or BMP-4 transiently co-expressed with BMP-7, resulted in more BMP activity than expression of any single BMP. Stable cell lines were then made in order to purify and characterize co-expressed BMPs in more detail. Co-expression of BMP-2 with BMP-7 yielded heterodimeric BMP-2/7 with a specific activity about 20-fold higher than BMP homodimers in an in vitro alkaline phosphatase induction assay. These heterodimers were also 5- to 10-fold more potent than BMP-2 in inducing cartilage and bone in an in vivo assay. Similar results were obtained with BMP-2/6 heterodimer. These experiments demonstrate the increased potency of several BMP heterodimers relative to BMP homodimers and support the hypothesis that such heterodimeric forms are likely to have natural biological functions.

The BMP proteins in bone formation and repair

From recent advances in the fields of bone biology and pattern formation, the first clues to our understanding of embryonic skeletal development are beginning to emerge. This complex process involves an integration of spatial patterning and the differentiation of specialized cells that make up bone and cartilage. The result is a scale model of the mature skeleton which is able to grow in size to fit the adult body plan. In the mature animal, bone repair after injury appears to be similar to bone formation in the embryo, suggesting that analogous mechanisms for the control of bone formation may exist in the adult and embryonic skeletons.

The localized expression of extracellular matrix components in healing tendon insertion sites: an in situ hybridization study

The localized expression of a number of extracellular matrix genes was evaluated over time in a novel rat rotator cuff injury model. The supraspinatus tendons of rats were severed at the bony insertion and repaired surgically. The healing response was evaluated at 1, 2, 4, and 8 weeks post‐injury using histologic and in situ hybridization techniques. Expression patterns of collagens (I, II, III, IX, X, XII), proteoglycans (decorin, aggrecan, versican, biglycan, fibromodulin), and other extracellular matrix proteins (elastin, osteocalcin, alkaline phosphatase) were evaluated at the healing tendon to bone insertion site. Histologic results indicate a poor healing response to the injury, with only partial recreation of the insertion site by 8 weeks. In situ hybridization results indicate a specific pattern of genes expressed in each zone of the insertion site (i.e., tendon, fibrocartilage, mineralized cartilage, bone). Overall, expression of collagen types I and XII, aggrecan, and biglycan was increased, while expression of collagen type X and decorin was decreased. Expression of collagen type I, collagen type XII, and biglycan decreased over time, but remained above normal at 8 weeks. Results indicate that the rat supraspinatus tendon is ineffective in recreating the original insertion site, even at 8 weeks post‐injury, in the absence of biological or biomechanical enhancements.

BMP2 signaling in bone development and repair.

BMPs are best known for their actions as bone formation signals. Recent studies using transgenic mice in which individual osteogenic BMPs have been removed from the limb skeleton have identified BMP2 as a fundamental component of the inherent regenerative capacity of bone. This review summarizes current findings on the specific requirement for BMP2 in bone formation and repair.

BMP and BMP inhibitors in bone.

Abstract:  Bone morphogenetic proteins (BMPs) are signaling molecules that act locally on target cells to affect cell survival, proliferation, and differentiation. While first identified as bone‐inducing agents, BMPs are now known to affect the formation and function of many organ systems. Here we focus only on the roles of BMPs in the skeleton. In the developing mouse embryo, BMPs direct skeletal patterning, chondrogenesis, and bone formation. In postnatal animals, BMPs are potent bone regeneration factors, affecting both the amount of new bone formed and the rate at which bone healing occurs. The amount of BMP available for signaling is tightly regulated in both the embryo and postnatally, and in the context of the skeleton, several structurally distinct BMP ligand antagonists have been shown to alter the ability of BMPs to bind to their receptors, blocking BMP activity in physiologically important circumstances. For example, noggin knockout mice display cartilage hyperplasia during skeletal development that results in the loss of joint formation (too much BMP activity), while mice that overexpress noggin in skeletal cells display severe osteopenia and bone fragility (too little BMP activity). Sclerostin, chordin, CTGF, follistatin, and gremlin are additional BMP antagonists that may act in the skeleton to regulate BMP availability. Another class of BMP inhibitors are the proteins that bind to BMP receptors but have no inherent signaling function and thus act as BMP receptor antagonists. To date, inhibin and BMP‐3 have been identified as BMP receptor antagonists that can block BMP signaling in bone. Identification of BMP antagonists allows us to investigate their role in diseases that affect skeletal function, such as osteopenia and nonunion fracture, and may provide a novel therapeutic intervention point for treatment.