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.     

Extracellular bone matrix-derived growth factor

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.     

Chondroclasts in osteoneogenesis

Allogenic, demineralized bone powder (DBP) was implanted into rat rectus abdominis muscle to induce osteoneogenesis. The main induction steps are invasion of the implant by host mesenchyme cells, differentiation of cartilage, invasion by blood capillaries or angiogenesis, differentiation of osteoblasts and bone marrow. The result is the formation of a cancellous ossicle. Giant polykarions appear in the implant after calcification of the cartilage matrix. As the DBP particles are not resorbed in the implant, these polykarions could either be foreign body giant cells brought about in reaction to foreign matrix or chondroclasts which resorb the cartilage. The results obtained by histological and histochemical methods (McNeals-von Kossa stain, tartrate resistant acid phosphatase reaction), as well as ultrastructural studies, lead to the conclusion that these large polynucleated cells are chondroclasts.     

The bone morphogenetic protein family and osteogenesis.

The BMPs (bone morphogenetic proteins) are a group of related proteins originally identified by their presence in bone-inductive extracts of demineralized bone. By molecular cloning, at least six related members of this family have been identified and are called BMP-2 through BMP-7. These molecules are part of the TGF-beta superfamily, based on primary amino acid sequence homology, including the absolute conservation of seven cysteine residues between the TGF-betas and the BMPs. The BMPs can be divided into subgroups with BMP-2 and BMP-4 being 92% identical, and BMP-5, BMP-6, and BMP-7 being an average of about 90% identical. To examine the individual activities of these molecules, we are producing each BMP in a mammalian expression system. In this system, each BMP is synthesized as a precursor peptide, which is glycosylated, processed to the mature peptide, and secreted as a homodimer. These reagents have been used to demonstrate that single molecules, such as BMP-2, are capable of inducing the formation of new cartilage and bone when implanted ectopically in a rodent assay system. Whether each of the BMPs possesses the same inductive activities in an animal is the subject of ongoing research. Based on the chondrogenic and osteogenic abilities of the BMPs in the adult animal, the expression of the mRNAs for the BMPs has been examined in the development of the embryonic skeleton by in situ hybridization. These studies demonstrate that the BMP mRNAs are spatially and temporally expressed appropriately for the proteins involved in the induction and development of cartilage and bone in the embryonic limb bud.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of cartilage by non-chondrogenic cell types

Freshly excised embryonic rat skeletal muscle has been shown to form hyaline cartilage when organ cultured upon demineralized rat bone (bone matrix). Since skeletal muscle is composed of fibrous connective tissue (C.T.) as well as muscle cells, the cartilage could arise from either of these sources. The object of this study was to determine whether cartilage arose from fibrous connective tissue or muscle cells, or both, and whether the ability to form cartilage is limited to tissues derived from somatic mesoderm. Control experiments demonstrated that 19-day embryonic rat skeletal muscle formed cartilage when organ cultured on bone matrix after dissociation and cultivation in vitro, and that 11-day embryonic chick muscle also formed cartilage, although less reproducibly (3 out of 10 cases). Fibroblasts and skeletal muscle were cloned from similar suspensions of dissociated muscle in order to test these purified cell types. Dermis, vascular tissue, and tendons were mechanically removed prior to dissociation in order to eliminate fibroblasts from contaminant sources. Cloned fibroblasts, derived from rat skeletal muscle, formed cartilage in three out of three cases. It was not possible to clone sufficient rat skeletal muscle to place an aggregate onto bone matrix. An aggregate of several hundred chick skeletal muscle clones formed cartilage on bone matrix. The freshly excised C.T. capsules of embryonic chick thyroid and lung were tested for the ability to form cartilage as nonskeletal C.T. derivatives. The epithelial rudiments of thyroid and lung were also tested as endodermal derivatives. Chick cornea was similarly tested as an ectodermal derivative. Of these tissues, only the C.T. capsules formed cartilage. The results demonstrate that various C.T. cell types may alter their phenotype well after that stage at which their differentiation is thought to be stabilized, and that the ability to differentiate as cartilage may be common to all C.T. cells. The option of differentiating along a certain variety of pathways may depend more upon local conditions than on a predetermined pattern.     

Persistence of cartilage proteoglycan and link protein during matrix-induced endochondral bone development: An immunofluorescent study

Monospecific antibodies to cartilage proteoglycan monomer and link protein were employed with immunofluorescence microscopy to determine the tissue distribution of these constituents during matrix-induced endochondral bone development. Subcutaneous implantation of demineralized diaphyseal bone matrix resulted in new endochondral bone formation. On Day 3, the implant consisted of mesenchymal tissue which did not contain any demonstrable cartilage-related proteoglycan or link protein. With the onset of early chondrogenesis on Day 5, cartilage proteoglycan monomer and link protein were first localized together in the cartilage matrix, particularly around chondrocytes in territorial sites. Progressively more staining around cells was observed at Days 7 and 9. On Day 9, when mineralization was first observed, there was no evidence of a net loss of these molecules prior to mineralization of the cartilage matrix. On Day 11 and thereafter, bone formation was observed by appositional growth on calcified cartilage spicules. Whereas the osteoblasts and bone matrix were devoid of any staining for cartilage proteoglycan and link components, the residual, partly mineralized cartilage spicules still reacted with antibodies to cartilage proteoglycan monomer and link protein in territorial sites, but in reduced amounts, indicating a loss of these molecules associated with a loss of hypertrophic chondrocytes. Since mineral prevented the access of Fab' antibody subunits, demineralization after fixation was routinely employed. The results reveal that cartilage proteoglycan monomer and link protein are present around chondrocytes in hyaline cartilage during the early stages of endochondral bone formation and that there is no net loss of these molecules prior to mineralization of this cartilage matrix as was previously thought.     

Changes in ornithine decarboxylase activity during matrix-induced cartilage, bone and bone marrow differentiation.

Subcutaneous transplantation of coarse powders of demineralized rat diaphyseal bone matrix into allogeneic recipients results in new bone formation. The changes in ornithine decarboxylase activity during such bone matrix-induced sequential differentiation of cartilage, bone and bone marrow were investigated. There was a peak in ornithine decarboxylase activity on day 3 corresponding to the appearance of fibroblasts in close contiguity to the bone matrix. This was followed by another peak of enzyme activity on day 8 which was correlated with the onset of proliferation of presumptive osteoblasts and vascular endothelial cells. The peak of ornithine decarboxylase activity on day 3 appears to be a demineralized bone matrix-specific event. Induction of ornithine decarboxylase activity represents one of the early responses to implanted bone matrix.     

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.     

An experimental analysis of factors affecting the localization of embryonic bone marrow

Summary The present investigations have been concerned with factors which determine and influence the localization and development of hemopoietic bone marrow in the embryo mouse and the adult. These studies, which have employed organ cultures and the transplantation of mouse embryo femur and tail rudiments, indicate that the surrounding mesenchyme is required for the normal development of the cartilage rudiment and its ossification, and for the formation and colonization of the marrow cavity. It was suggested that hemopoiesis results from the colonization of the “prepared” marrow cavity by stem cells arising from sources external to the rudiment. The addition of erythropoietin and L-thyroxine produced distinct erythropoietic differentiation in the normally myelocytic embryonic marrow cavity. The significance of the microenvironment present in developing bone rudiments and the initiation of hemopoiesis in stem cells was discussed. A hypothesis was developed to explain marrow localization in adults based on the colonization of bone rudiments which are developing their marrow sites at a time when the blood contains large numbers of colony-forming units.     

Chondrogenesis in agarose gel culture : A model for chondrogenic induction, proliferation and differentiation

An in vitro model has been developed to study chondrogenic induction, proliferation and differentiation. Embryonic rat mesenchymal cells isolated from muscle and embedded in agarose were treated with a partially purified extract from bovine demineralized bone powder. Treated cells proliferated and synthesized matrix similar to differentiated chondrogenic cells in a dose-dependent manner. By employing an enzyme-linked immunosorbent assay (ELISA), cartilage-specific proteoglycan and type II collagen synthesis were quantitated. Of the cells tested, only embryonic mesenchymal cells from muscle responded to bone extract. Proteoglycan synthesis was sensitive to type of medium and cell density.     

Induction of heterotopic and orthotopic cartilage and bone formation in mice

Heterotopic cartilage, bone and bone-marrow formation was achieved in mice by transplantation of a variety of xenogeneic established cell lines, by the transitional epithelium or by implants of demineralized bone matrix. The pattern and the sequence of events were always the same, regardless of the inducer used; viz., hyaline cartilage appeared 6-7 days after implantation, and endochondral bone formation followed. However, in cases of allogeneic implants of transitional epithelium into species other than the mouse, an intramembranous osteogenesis was the main mode of bone formation. When the yield of induced bone was high enough, a true myelopoiesis developed after three weeks. Heterotopically-induced bones had a relatively short life-span. Periosteal membranes of bones at the sites of sarcomes induced by M-MSV responded with rapid and extensive proliferation, with subsequent bone and, sometimes, hyaline cartilage deposition. This phenomenon was observed in long and cranial bones. However, bone induced heterotopically by demineralized bone matrix did not respond in such a way to the presence of M-MSV-induced sarcoma, suggesting that the connective tissue-encapsulated heterotopic bone was not a functioning periosteum. M-MSV-induced sarcoma also stimulates proliferation of elastic cartilage.     

Dissociative extraction and partial purification of osteogenin, a bone inductive protein, from rat tooth matrix by heparin affinity chromatography

Implantation of demineralized tooth matrix in subcutaneous sites results in new bone formation locally. The osteoinductive activity of the tooth matrix was dissociatively extracted in 4.0 M guanidine hydrochloride and the residue was devoid of biologic activity. The bone inductive protein, osteogenin, was partially purified by heparin affinity chromatography. The heparin binding fraction initiated the bone differentiation cascade when implanted with guanidine extracted, inactive bone or tooth matrices. These results imply a cooperative interaction between the soluble osteogenin and collagenous substratum in bone induction.     

Osteogenin (bone morphogenetic protein-3) stimulates cartilage formation by chick limb bud cells in vitro.

Osteogenin is a protein isolated from demineralized bovine bone matrix. When implanted in rats, osteogenin induces the differentiation of cartilage and formation of endochondral bone. When added to stage 24 and 25 chick limb bud mesoderm cells in culture, it stimulated synthesis of sulfated proteoglycans by over 10-fold without stimulating cell division. The increase was detected after only 2 days in culture. Morphologically, in the presence of osteogenin, all cells in the culture appeared to form cartilage, rather than the nodules of cartilage surrounded by noncartilage areas in control cultures. The distribution of type II collagen correlated with the morphological differentiation of cartilage. When nonchondrocyte and chondrocyte cell populations were separated, osteogenin stimulated sulfated proteoglycan synthesis in all populations of cells. However, the greatest stimulation (24-fold) was seen in the originally nonchondrocyte population, which apparently still had some potential to form cartilage. In this study, chick limb bud mesoderm cells in vitro responded to osteogenin, a protein derived from adult bovine bone matrix. The cells that were responsive included those that initially did not form cartilage. Osteogenin belongs to a superfamily of proteins, many of which are important in development. It is possible that osteogenin has a role in embryonic cartilage development.     

Accumulation, localization, and compartmentation of transforming growth factor beta during endochondral bone development

Endochondral bone formation was induced in postnatal rats by implantation of demineralized rat bone matrix. Corresponding control tissue was generated by implanting inactive extracted bone matrix, which did not induce bone formation. At various times, implants were removed and sequentially extracted with guanidine hydrochloride, and then EDTA and guanidine hydrochloride. Transforming growth factor beta (TGF beta) in the extracts was quantitated by a radioreceptor assay. TGF beta was present in demineralized bone matrix before implantation, and the concentration had decreased by 1 d after implantation. Thereafter, TGF beta was undetectable by radioreceptor assay until day 9. From day 9-21 the TGF beta was extracted only after EDTA demineralization, indicating tight association with the mineralized matrix. During this time, the content of TGF beta per milligram soluble protein rose steadily and remained high through day 21. This increased concentration correlated with the onset of vascularization and calcification of cartilage. TGF beta was detected only between days 3-9 in the controls; i.e., non-bone-forming implants. Immunolocalization of TGF beta in bone-forming implants revealed staining of inflammatory cells at early times, followed later by staining of chondrocytes in calcifying cartilage and staining of osteoblasts. The most intense staining of TGF beta was found in calcified cartilage and mineralized bone matrix, again indicating preferential compartmentalization of TGF beta in the mineral phase. In contrast to the delayed expression of TGF beta protein, northern blot analysis showed TGF beta mRNA in implants throughout the sequence of bone formation. The time-dependent accumulation of TGF beta when cartilage is being replaced by bone in this in vivo model of bone formation suggests that TGF beta may play a role in the regulation of ossification during endochondral bone development.     

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.     

Appearance of fibronectin during the differentiation of cartilage, bone, and bone marrow

Fibronectin has been localized by indirect immunofluorescence during the various phases of endochondral bone formation in response to subcutaneously implanted demineralized bone matrix. Its histologic appearance has been correlated with results of biosynthetic experiments. (a) The implanted collagenous bone matrix was coated with fibronectin before and during mesenchymal cell proliferation. (b) During proliferation of mesenchymal precursor cells, the newly synthesized extracellular matrix exhibited a fibrillar network of fibronectin. (c) During cartilage differentiation, the fibronectin in the extracellular matrix was apparently masked by proteoglycans, as judged by hyaluronidase treatment. (d) Differentiating chondrocytes exhibited a uniform distribution of fibronectin. (e) Fibronectin was present in a cottony array around osteoblasts during osteogenesis. (f) The developing hematopoietic colonies revealed fibronectin associated with them. Therefore, it appears that fibronectin is ubiquitous throughout the development of endochondral bone and bone marrow.     

Changes in the gene expression of collagens, fibronectin, integrin and proteoglycans during matrix-induced bone morphogenesis

Subcutaneous implantation of demineralized bone matrix in rat results in the local cartilage and bone development. This in vivo model of bone formation was used to examine the expression patterns of cartilage and bone specific extracellular matrix genes. The steady state levels of mRNA in implants for cartilage specific type II collagen, type IX collagen, proteoglycan link protein and cartilage proteoglycan core protein (aggrecan) were increased during chondrogenesis and cartilage hypertrophy. Fibronectin mRNA levels were high during mesenchymal cell migration, attachment and chondrogenesis. Integrin (beta 1 chain) mRNA was expressed throughout the endochondral bone development. Type I collagen mRNA levels in implants increased as early as day 3, reached its peak during osteogenesis. These gene markers will be useful in the study of the mechanism of action of bone morphogenetic proteins present in the demineralized bone matrix.     

调控软骨形成的信号通路及相关因子在骨髓间充质干细胞骨向分化中的作用*

骨髓间充质干细胞是一类具有自我复制和多向分化潜能的成体干细胞,可以通过定向诱导分化为成骨细胞、软骨细胞、脂肪细胞等,是目前骨再生医学和细胞治疗研究最多的理想种子细胞。在骨缺损的修复过程中,骨髓间充质干细胞内成软骨相关基因表达升高进而分化为软骨细胞,后期随着成骨细胞和破骨细胞的形成及血管长入,软骨基质逐步降解并被骨基质所替换。软骨细胞参与了骨缺损前期的修复过程,调控软骨形成的信号通路及相关因子不仅调控骨髓间充质干细胞成软骨细胞分化,同时在成骨细胞分化过程中也发挥着重要的作用。对调控软骨形成的信号通路及相关因子在骨髓间充质干细胞骨向分化中的调控作用和研究现状进行了总结,以期为临床寻找更好的治疗骨缺损的方法提供理论依据和研究方向。     

A water-soluble fraction from adult bone stimulates the differentiation of cartilage in explants of embryonic muscle

A water-soluble fraction of a 4 M guanidine HCl extract of demineralized adult bovine bone stimulated the differentiation of cartilage in explants of minced skeletal muscle from embryonic chick legs; cartilage was also induced by a semipurified protein preparation. Cartilage could be identified in treated cultures at 1 week with muscle from day-9 embryos, not before 2 weeks with muscle from day-12 embryos, and not before 3 weeks with muscle from day-19 embryos. The ability to respond to this water-soluble fraction by exhibiting cartilage differentiation was dose-dependent, but not confined to any particular muscle region of the day-12 embryonic leg. These observations indicate that bone-derived soluble chondroinductive agents act on cells in minced embryonic muscle preparations. The induction of cartilage is dependent upon the accessibility of the responding cells to the agents, on the concentration of inductive agents, and on the developmental age of the responsive tissue.