Noreen S. Cunningham

Initiation of bone regeneration in adult baboons by osteogenin, a bone morphogenetic protein

Osteogenin, and related bone morphogenetic proteins, induce endochondral bone differentiation through a cascade of events which include formation of cartilage, hypertrophy and calcification of the cartilage, vascular invasion, differentiation of osteoblasts, and formation of bone. These events have been studied in a postnatal model of bone development in rodents. Information concerning the morphogenetic potential of osteogenin in primates is a prerequisite for potential clinical application in man. The efficacy of allogeneic osteogenin in primates was investigated in both extraskeletal and skeletal sites in 19-Chacma baboons (Papio ursinus). Osteogenin was isolated from demineralized baboon bone matrix and purified by chromatography on heparin-Sepharose, hydroxyapatite, and Sephacryl S-200. Protein fractions with a molecular mass range of 26-42 kDa induced cartilage and bone differentiation in the subcutaneous space of rats. Final purification to homogeneity was obtained by electroendosmotic elution from a preparative sodium dodecyl sulphate (SDS) polyacrylamide gel, resulting in a single band on a SDS-polyacrylamide gel with an apparent molecular mass of 30-34 kDa, with biological activity in rats. The osteoinductive potential of osteogenin in primates was tested first in intramuscular sites in baboons and found to be active. The bone regeneration potential was investigated in nonhealing calvarial defects surgically prepared in adult male baboons. Baboon osteogenin induced complete regeneration of the cranial wound. These findings in adult primates establish a primary role for osteogenin in initiation and promotion of osteogenesis, and imply a potential therapeutic application based on cell biology of extracellular matrix-cell interactions.

Tricalcium phosphate and osteogenin: A bioactive onlay bone graft substitute

The disadvantages of autogenous bone grafts has prompted a search for a dependable onlay bone graft substitute. A combination of tricalcium phosphate, a resorbable ceramic, and osteogenin, an osteoinductive protein, was evaluated as an onlay bone graft substitute in a rabbit calvarial model. Twenty-eight tricalcium phosphate implants (15 mm diameter x 5 mm; pore size, 100-200 microns) were divided into experimental and control groups and placed on the frontal bone of 14 adult New Zealand White rabbits. In the experimental animals, 185 micrograms of osteogenin was added to each implant. In the control animals, the implants were placed untreated. Implants were harvested at intervals of 1, 3, and 6 months, and evaluated using hematoxylin and eosin histology, microradiography, and histomorphometric scanning electron microscope backscatter image analysis. At 1 month there was minimal bone ingrowth and little tricalcium phosphate resorption in both the osteogenin-treated and control implants. At 3 months, both the osteogenin-treated and control implants showed a modest increase in bone ingrowth (8.85 percent versus 5.87 percent) and decrease in tricalcium phosphate (32.86 percent versus 37.08 percent). At 6 months, however, the osteogenin-treated implants showed a statistically significant increase in bone ingrowth (22.33 percent versus 6.96 percent; p = 0.000) and decrease in tricalcium phosphate (27.25 percent versus 37.80 percent; p = 0.004) compared with the control implants. The bone within the control implants was mostly woven at 6 months, whereas the osteogenin-treated implants contained predominantly mature lamellar bone with well-differentiated marrow. All implants maintained their original volume at each time interval studied. The tricalcium phosphate/osteogenin composite, having the advantage of maintaining its volume and being replaced by new bone as the tricalcium phosphate resorbs, may be applicable clinically as an onlay bone graft substitute.

Autoradiographic localization of osteogenin binding sites in cartilage and bone during rat embryonic development.

Osteogenin, a novel bone differentiation factor isolated from bone, has been recently purified and the amino acid sequence determined. Osteogenin in conjunction with a collagenous bone matrix substratum induces cartilage and bone formation in vivo. In order to understand the developmental role of osteogenin during cartilage and bone morphogenesis we examined the binding and distribution of iodinated osteogenin in developing rat embryos. Whole embryo tissue sections were made from 11, 12, 13, 15, 18, and 20 day fetuses. The specific binding of osteogenin at different stages of rat embryonic development was determined by autoradiography. Maximal binding was observed in mesodermal tissues such as cartilage, bone, perichondrium, and periosteum. During Days 11-15, peak binding was localized to perichondrium during limb and vertebral morphogenesis. By Day 18 periosteum exhibited the highest concentration of autoradiographic grains. Osteogenin was also localized in developing membranous bones of the calvarium and other craniofacial bones. Considerably less binding was observed, in decreasing order, in muscle, liver, spleen, skin, brain, heart, kidney, and intestine. The observed maximal binding during skeletal morphogenesis implies a developmental role for osteogenin.

Initiation of bone development by osteogenin and promotion by growth factors

The cellular and molecular basis of bone development and its regulation by differentiation and growth factors is an exciting area of current research. This article briefly reviews the historical progress in the isolation of osteogenin, a novel bone differentiation factor, and its modulation by well known growth factors. Endochondral bone development is a multistep sequential cascade and the process must be operationally dissected. It has been accomplished with the demineralized bone matrix-induced bone formation model. The reproducible development of cartilage and bone in an extraskeletal site permits the study of the initiation of the first cycle of endochondral bone formation and mineralization. Recent progress in the isolation of osteogenin, a specific bone differentiation factor, by heparin affinity chromatography permits the further investigation of the commitment and clonal expansion of the putative osteoprogenitor stem cells. Once initiated, bone formation is promoted by growth factors such as platelet derived growth factor, fibroblast growth factor, insulin like growth factor, transforming growth factor beta and a plethora of non specific cytokines. Finally bone development is further modulated by systemic hormones and nutrition and a host of physical signals including electrical, gravitational and mechanical forces.

Recent Progress in Bone Induction by Osteogenin and Bone Morphogenetic Proteins: Challenges for Biomechanical and Tissue Engineering

Implantation of demineralized bone matrix results in local bone induction. Bone induction is a sequential biological chain reaction that consists of chemotaxis and proliferation of mesenchymal cells and differentiation of bone. Osteogenin, a bone morphogenetic protein has been purified and the amino acid sequence determined. Recently a family of bone morphogenetic proteins have been cloned and expressed by recombinant DNA technology. The availability of growth and morphogenetic factors will permit the rational design of new bone. The challenge for the biomechanical engineer is to attain mechanically optimal and functionally adaptive new bone for various skeletal prostheses. We are on the threshold for fabrication of new bone based on sound architectural design principles of tissue engineering based on cellular and molecular biology of growth and differentiation factors.