T K Sampath

Distinct and overlapping patterns of localization of bone morphogenetic protein (BMP) family members and a BMP type II receptor during fracture healing in rats

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) are thought to play an important role in bone morphogenesis. The purpose of this study was to determine the locations of BMP-2/-4, osteogenic protein-1 (OP-1, also termed BMP-7), and BMP type II receptor (BMPR-II) during rat fracture healing by immunostaining, and thereby elucidate the possible roles of the BMPs and BMPR-II in intramembranous ossification and endochondral ossification. In the early stage of fracture repair, the expression of BMP-2/-4 and OP-1 was strongly induced in the thickened periosteum near the fracture ends, and coincided with an enhanced expression of BMPR-II. On day 7 after fracture, staining for BMP-2/-4 and OP-1 immunostaining was increased in various types of chondrocytes, and was strong in fibroblast-like spindle cells and proliferating chondrocytes in endochondral bone. On day 14 after fracture, staining with OP-1 antibody disappeared in proliferating and mature chondrocytes, while BMP-2/-4 staining continued in various types of chondrocytes until the late stage. In the newly formed trabecular bone, BMP-2/-4 and OP-1 were present at various levels. BMPR-II was actively expressed in both intramembranous ossification and endochondral ossification. Additionally, immunostaining for BMP-2/-4 and OP-1 was observed in multinucleated osteoclast-like cells on the newly formed trabecular bone, along with BMPR-II. In reference to our previous study of BMP type I receptors (BMPR-IA and BMPR-IB), BMPR-II was found to be co-localized with BMPR-IA and BMPR-IB. BMP-2/-4 and OP-1 antibodies exhibited distinct and overlapping immunostaining patterns during fracture repair. OP-1 may act predominantly in the initial phase of endochondral ossification, while BMP-2/-4 acts throughout this process. Thus, these findings suggested that BMPs acting through their BMP receptors may play major roles in modulating the sequential events leading to bone formation.

Expression and localization of bone morphogenetic proteins (BMPs) and BMP receptors in ossification of the ligamentum flavum

To clarify the pathogenesis of ossification of the ligamentum flavum (OLF), we examined the expression and localization of bone morphogenetic proteins (BMPs) and their receptors (BMPRs) in the ligamentum flavum of the patients with OLF by immunohistochemical staining and compared them with staining patterns in control patients. The BMPRs appeared extensively in mature and immature chondrocytes around the calcified zone and in spindle-shaped cells and round cells in the remote part from ossified foci in examined tissue of OLF. The ligands for BMPRs, BMP-2/-4 and osteogenic protein-1 (OP-1)/BMP-7, colocalized in OLF patients. In the control cases, expression of BMPs and BMPRs was observed around the calcified zone at the insertion of the ligamentum flavum to the bone, and limited expression was found in the smaller range. Thus, the expression profile of BMPs and BMPRs in OLF patients was entirely different from the control patients, suggesting that BMPs may be involved in promoting endochondral ossification at ectopic ossification sites in OLF, and that ossification activity is continuous in these patients.

Long-Term Evaluation of Bone Formation by Osteogenic Protein 1 in the Baboon and Relative Efficacy of Bone- Derived Bone Morphogenetic Proteins Delivered by Irradiated Xenogeneic Collagenous Matrices

To investigate the long‐term efficacy of irradiated recombinant human osteogenic protein 1 (hOP‐1) in bone regeneration and morphogenesis, hOP‐1 was combined with a bovine collagenous matrix carrier (0, 0.1, 0.5, and 2.5 mg hOP‐1/g of matrix), sterilized with 2.5 Mrads of γ‐irradiation, and implanted in 80 calvarial defects in 20 adult baboons (Papio ursinus). The relative efficacy of partially purified bone‐derived baboon bone morphogenetic proteins (BMPs), known to contain several osteogenic proteins, was compared with the recombinant hOP‐1 device in an additional four baboons. Histology and histomorphometry on serial undecalcified sections prepared from the specimens harvested on day 90 and day 365 showed that γ‐irradiated hOP‐1 devices induced regeneration of the calvarial defects by day 90, although with reduced bone area compared with a previous published series of calvarial defects treated with nonirradiated hOP‐1 devices. One year after application of the irradiated hOP‐1 devices, bone and osteoid volumes and generated bone tissue areas were comparable with nonirradiated hOP‐1 specimens. Moreover, 365 days after healing regenerates induced by 0.5 mg and 2.5 mg of irradiated hOP‐1 devices showed greater amounts of bone and osteoid volumes when compared with those induced by nonirradiated hOP‐1 devices. On day 90, defects treated with 0.1 mg and 0.5 mg of bone‐derived baboon BMPs, combined with irradiated matrix, showed significantly less bone compared with defects receiving irradiated devices containing 0.1 mg and 0.5 mg hOP‐1; 2.5 mg of partially purified BMPs induced bone and osteoid volumes comparable with the 0.1‐mg and 0.5‐mg hOP‐1 devices. Control specimens of γ‐irradiated collagenous matrix without hOP‐1 displayed a nearly 2‐fold reduction in osteoconductive bone repair when compared with nonirradiated controls. These findings suggest that the reduction in bone volume and bone tissue area on day 90 may be caused by a reduced performance of the irradiated collagenous matrix substratum rather than to a reduction in the biological activity of the irradiated recombinant osteogenic protein. This is supported by the results of in vitro and in vivo studies performed to determine the structural integrity of the recovered γ‐irradiated hOP‐1 before application in the baboon. Recoveries by high‐performance liquid chromatography (HPLC) and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE)/immunoblot analyses indicated that doses of 2.5‐3 Mrads of γ‐irradiation did not significantly affect the structural integrity of the recovered hOP‐1. Biological activity of the recovered hOP‐1 was confirmed in vitro by showing induction of alkaline phosphatase activity in rat osteosarcoma cells (ROS) and in vivo by de novo endochondral bone formation in the subcutaneous space of the rat. These findings in the adult primate indicate that a single application of γ‐irradiated hOP‐1 combined with the irradiated xenogeneic bovine collagenous matrix carrier is effective in regenerating and maintaining the architecture of the induced bone at doses of 0.5 mg/g and 2.5 mg/g of carrier matrix.

Extracellular bone matrix-derived growth factor

Abstract Demineralized extracellular bone matrix, when implanted subcutaneously into allogeneic rats, induces an invariant sequence of events resulting in de novo cartilage, bone and bone marrow formation. We have recently demonstrated the dissociative extraction and successful biological reconstitution of boneinducing molecule(s) in demineralized bone matrix. As mesenchymal cell proliferation precedes differentiation of endochondral bone, we have examined the bone-inductive molecules for mitogenic activity on human and rat fibroblasts and bovine endothelial cells. The results revealed that the molecular fraction obtained by molecular sieve chromatography on Sepharose CL-6B responsible for endochondral bone induction is capable of stimulating human and rat fibroblasts proliferation by 2500 and 300 % respectively, as compared to cells grown in a serum-free medium. Endothelial cells do not seem to share this response. This factor exhibits a significant effect on growth-promoting activity for both human and rat fibroblasts at a dose of 0.5 ng protein/ml of culture medium. These results demonstrate the presence of a tightly bound growth factor in the extracellular bone matrix.

Distribution of bone inductive proteins in mineralized and demineralized extracellular matrix.

Implantation of demineralized extracellular bone matrix results in new bone formation locally. Although the precise molecular mechanisms are not known, the reconstitution of matrix proteins less than 50,000 daltons with collagenous residue results in bone induction. The aim of the present investigation was to ascertain the distribution of the bone inductive protein(s) in various compartments of the tissue. A sequential extraction of mineralized bone matrix was employed: (1) 4 M guanidine HCl to extract proteins that are cell associated and not masked by mineral; (2) 0.5 M EDTA to dissolve the mineral phase; (3) 4 M guanidine HCl to reextract the collagenous matrix-associated proteins under dissociative conditions; (4) 4 M guanidine HCl containing 0.5 M EDTA to release any other residual proteins. This sequential method revealed that about 25% of total biological activity of bone induction is associated with first guanidine extraction, about 15% with the mineral phase and the rest of the activity is tightly associated with the collagenous matrix.

Bone morphogenetic protein type IB receptor is progressively expressed in malignant glioma tumours.

The distribution of bone morphogenetic protein (BMP) type I receptors and the activin type I receptor (ActR-I) was investigated in 16 cases of human glioma and five cases of non-tumourous gliosis tissue by immunohistochemical technique. Both BMP type IA (BMPR-IA) and the type IB (BMPR-IB) receptors were detected in human glioma cells. A significant increase in BMPR-IB in tumour cells was observed in malignant glioma compared with both low-grade astrocytomas (n=16, P<0.005) and gliosis (n=13, P<0.001). However, enhancement of BMPR-IA staining was moderate and ActR-I staining was only weakly expressed in the malignant glioma tumours. Osteogenic protein (OP)-1/BMP-7, which is known to bind BMPR-IA, BMPR-IB and ActR-I, was expressed in nervous tissue and was also detected in anaplastic areas of malignant glioma. In contrast to the tissue materials, BMPR-IA was expressed to a stronger degree than BMPR-IB in human glioma cell lines; the growth of these cells was suppressed by OP-1. These results suggest the presence of BMP receptors and a functional role for BMPs in malignant glioma.

Extracellular matrix proteins involved in bone induction are vitamin D dependent

Subcutaneous implantation of demineralized diaphyseal bone matrix into allogeneic rats results in local formation of cartilage and bone. However, implantation of demineralized bone matrix obtained from rachitic rats did not induce bone. Rachitic bone matrix was therefore dissociatively extracted with 4 M guanidine HCl and then reconstituted with an inactive collagenous residue of control as carrier. Such reconstituted materials also lacked bone inductive potential. On the other hand, reconstitution of guanidine HCl extracts of control bone matrix with inactive vitamin D deficient matrix did result in bone induction. Partial purification (fractions containing proteins (less than 50,000 daltons) of the guanidine HCl extract from rachitic rats on Sepharose CL-6B followed by reconstitution with inactive collagenous residues resulted in a weak (25% of control) inductive response. These observations imply that bone inductive proteins are vitamin D dependent and are reduced in matrix obtained from rachitic rats.

Comparison of bone inductive proteins of rat and porcine bone matrix

Subcutaneous implantation of demineralized bone matrix in allogenic rats induces a sequence of events resulting in de novo formation of cartilage, bone and bone marrow. In the present study endochondral bone formation by demineralized porcine matrix was studied and compared with the rat bone matrix. Endochondral bone formation was induced by 4M guanidine hydrochloride fraction IV (less than 50,000 daltons) of Sepharose CL-6B gel filtration but not by whole extract or by demineralized porcine bone matrix. Sephacryl S-200 gel filtration of the osteoinductive proteins of fraction IV showed the Porcine osteoinductive factor to be associated with protein fraction III (less than 20,000 daltons) whereas the rat with fraction II (between 20,000 and 30,000 daltons) of the chromatographic profile indicating an apparent difference in molecular weight of the osteoinductive factors between these two species.