Signaling Molecules

Bone tissue engineering offers the prospect of new therapies for clinically significant skeletal morbidity. Skeletal defects result from pathologic, traumatic, and developmental events. Whereas the etiology may differ, current treatment employs similar approaches for remediation. By contrast, bone tissue engineering provides customized therapies to augment and amplify the patient's clinical capability. Additional knowledge of the interrelationship of scaffold, growth factor, cell type and intracellular signaling mechanisms ensures further refinement of the bone tissue engineered construct. Scaffold integrity and geometry, growth factor affinity and targeting, identification of specific receptive cell types, and the mechanisms for intracellular signaling are among the emphasized at the Bone Tissue Engineering Center (BTEC).

Successful tissue engineering of bone is based on the molecular and biological knowledge of the cellular and subcellular characteristics of bone healing. The concurrent presence of cells, growth and differentiation factors, vascularity and a biomimetic scaffold are important to bone formation. These components may be incorporated into a therapeutic agent to enhance osteogenesis.

There are several strategies to achieve tissue regeneration. The simplest method is to supply growth factors to the site of regeneration for cell differentiation and proliferation. However, in general, direct injection of growth factor in solution into the regeneration site is ineffective, as the injected growth factor rapidly diffuses out from the site within one day. ·

• To remedy the rapid diffusion and enable the growth factor to efficiently exert the biological effects, techniques using drug delivery systems are employed. One such promising technique is the controlled release of growth factor at the site of action over a long time period by incorporating the factor into an appropriate carrier (bioabsorbable scaffolds). ·
• As an alternative to a growth factor, bioabsorbable scaffolds may be seeded with DNA coding for a growth factor. If a growth factor gene is transfected into the cells existing in the site of regeneration, the cell secretion of the growth factor, may promote tissue regeneration. ·
• Isolation of cells from the recipient, typically from bone marrow or connective tissue and expand them in tissue culture. These cells may be exposed to bioactive factors, combined with the scaffold and implanted into the donor site to promote regeneration. ·
• Development of cell lines (or stem cells) through gene therapy are engineered to synthesize and secrete specific growth factors, combined with a delivery system and implanted in the skeletal site. The cells will differentiate under the influence of the secreted growth factor.

Current work on suitable strategies for gene therapy makes the technology attractive for repairing defects in the musculoskeletal system. Delivering the genetic material into a targeted host cell can be accomplished by several techniques. Targeting vectors, either viral or nonviral, have been successfully applied for use either in situ or ex vivo and re-implanted. Ex vivo, an osteoblast or osteogenic precursor cell population can be altered genetically to express a preferred molecule, e.g. BMP-2 or BMP-4. Thereafter, the cells could be selected, expanded in number, and transplanted to a patient. Alternately, in situ delivery would involve the direct genetic manipulation of a host cell, either targeted or general. Targeted gene therapy is a rational and efficient alternative to methods that use exogenous growth factors.

Local gene therapy treatments may be options to compensate for a high-dose-for-effect of growth factor at an skeletal wound site. A collagen carrier containing DNA plasmids encoding human PTH1-34 and/or mouse BMP-4 have exhibited promising in situ delivery with pre-clinical results for regenerating skeletal defects. Several ex vivo approaches have included adenoviral gene transfer to generate BMP-2-producing bone-marrow cells. For these studies, critical-sized defects in rats were treated with genetically modified autogenous marrow cells delivered by allogeneic, inactivated demineralized bone matrix. The synthesized growth factors induced osteogenesis in the skeletal defects, however; concern for the virus' use is based upon the immunogenic potential of the virus.

Cell signaling involves the elucidation of both inter- and intra-cellular mechanisms. Alterations in cell activity and phenotype are means to observe the function of cell signaling. Histological, physical chemical, biochemical and molecular biological assessments of the cell are conducted to optimize conditions for a phenotype or activity.

The mechanism of growth factor control of cellular behavior depends on cell sensitivity to the growth factor. Following extracellular detection of growth factors cellular processes are initiated through intracellular signaling cascades. The diagram (Osteogenic Growth Factors) illustrates the numerous protein molecules that circulate through the extracellular enviroment adjacent to bone forming cells. The elucidation of signal transduction in bone cells has partially derived from the commonly shared pathways found in other cell types - muscle cells and fibroblasts. Several pathways are commonly found or suspected in all bone cell types and others currently appear to be unique to a phenotype.

Often, an intracellular signaling pathway is initiated with the binding of a substance to a receptor molecule. An intracellular signal may also be initiated by ion concentration gradients. Both mechanisms are found in the bone cells. By either mechanism, the signal requires the presence of an effector. Bone cells possess one or more effectors. Prominent effectors include tyrosine kinase linked effectors as well as ion channels, particularly divalent calcium, Ca++. Adenylate cyclase, guanylate cyclase, phospholipase A, C and D, and ion channels are G-protein linked effectors. The effectors are regulated through tyrosine phosphorylation by GTP-binding proteins.

Protein tyrosyl phosphorylation is an important element of cell development during proliferation and differentiation. Different groups of protein tyrosine kinases (PTK's) may participate in bone cell proliferation compared with differentiation. Two normal human bone cell types were employed to test the effect of two structurally different PTK's on cells in either a proliferative or differentiated state. Various PTK inhibitors were incubated with the cells. The different inhibitory capacity of each inhibitor on cells supported the involvement of PTKs in bone cell proliferation and differentiation. The differences in inhibitory capacity for each cell type suggested bone cell sensitivity to each inhibitor was due to the differences in cell state: proliferating compared with differentiated as determined by alkaline phosphatase assay.

The osteoblast, osteoclast, and osteocyte retain both commonly shared and lineage specific signal pathways. There may be temporal and spatial restrictions to the availability and use of intracellular signaling pathways. The pathways of intracellular signaling may be temporally restricted to a phase of bone cell development, require duration and concentration dependent threshold levels of exposure to the regulatory agent and depend upon the micro-environment around each cell.

The phenotype of each bone cell type remains controversial. The osteoblast is capable of synthesizing fibrillar type I collagen, secretes alkaline phosphatase, osteocalcin, osteonectin, osteopontin, and osteoprotegerin during the process of biomineralization. The osteoblast and the osteocyte are responsive to parathyroid hormone. The osteocyte descends from the osteoblast and is located within the bone to which it is uniquely adapted. Osteocytes appear to retain fewer cell organelles than osteoblasts. The osteocyte morphology facilitates communication through the canaliculi and endows them with their distinctive stellate morphology. Osteocytes possess a refined mechanosensory apparatus important to bone homeostasis. The specialized function and the decreased need to produce and secrete matrix may explain the reduced organelle content within the osteocyte. The osteoclast is a multinucleated giant cell associated with resorption lacunae and appears to lack many of the osteoblast receptors. Although not unique to the cell, osteoclast associated proteins include tartrate resistant acid phosphatase (TRAP), calcitonin, carbonic anhydrase II, a and b subunits of vitronectin receptors, and p60c-src receptors. Osteoclasts elaborate various proteolytic enzymes and create a micro-environment within which there is resorption of the mineralized matrix.

Based upon the premise that distinguishable cell types can be identified in the bone, we focus on osteoblast function within the skeletal wound site. The appreciation of the numerous interactions with other cells, the extracellular matrix and the potential conversion to an osteocyte is the context within which we study osteoblast activity. Similar considerations guide the design of a biomimetic, the scaffold, and the choice of protein factors to be included in the scaffold. The emphasis in these three areas: osteoblast, scaffold and protein factors are the central emphasis of the laboratory.