Activity and distribution of lysosomal enzymes during collagenous matrix-induced cartilage,bone, and bone marrow development

The appearance of the lysosomal enzymes acid phosphatase, arylsulfatase, and β-glucuronidase was studied during endochondral bone and bone marrow formation induced by implantation of demineralized bone matrix. The activities of acid phosphatase and β-glucuronidase gradually increased from the stage of mesenchymal cell proliferation on Day 3 onward to reach a peak on Day 13, during maximal bone remodeling. However, arysulfatase activity exhibited a sharp increase on Day 9, associated with the onset of cartilage hypertrophy and chondrolysis. The peak of arylsulfatase activity was also attained on Day 13. The activities of all three enzymes declined on Day 15 but acid phosphatase again exhibited an increase during hematopoietic bone marrow differentiation on Days 19–21. Histochemical and ultrastructural studies revealed intense lysosomal enzyme activity in macrophage-like cells on Day 7 and thereafter. During chondrolysis and bone remodeling, these cells were present in a perivascular location. Osteoclasts also exhibited strong reactivity for the lysosomal enzymes. Due to its characteristic temporal appearance during development of endochondral bone, arysulfatase may be used as a marker enzyme for chondrolysis and bone resorption.     

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 synthesis of types-I and -III collagen during matrix-induced endochondral bone differentiation in rat.

The changes in rates of hydroxyproline formation and biosynthesis of types-I and -III collagen during bone matrix-induced sequential differentiation of cartilage, bone and bone marrow in rat were investigated. Biosynthesis of types-I and -III collagen at different stages of this sequence was studied by labelling in vivo and in vitro with [2,3-3H]proline. Pepsin-solubilized collagens were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis. The results revealed that maximal amounts of type-III collagen were synthesized on day 3 during mesenchymal-cell proliferation. Thereafter, there was a gradual decline in type-III collagen synthesis. On days 9--20 during bone formation predominantly type-I collagen was synthesized. Similar results were obtained by the use of labelling techniques both in vivo and in vitro.     

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.     

Changes in tissue concentration of prostaglandins during endochondral bone differentiation

Prostaglandins are known to be involved in bone metabolism as evidenced by the ability of PGE2 to induce bone resorption. It was, therefore, of interest to determine if there was an association of specific prostaglandin metabolites with the various stages of developing bone by utilizing the matrix-induced endochondral bone formation system. During mesenchymal cell proliferation a peak of endogenous thromboxane B2 was detected. In the subsequent stages of chondrogenesis and chondrolysis PGF2 alpha was in high concentration, whereas during bone formation PGE2, 6-Keto-PGF1 alpha and thromboxane B2 were elevated. These changes in the peak levels of the various prostaglandin metabolites may reflect differences in the cell populations and function associated with various stages of endochondral bone formation.     

Time‐dependent contribution of BMP,FGF, IGF,and HH signaling to the proliferation of mesenchymal stroma cells during chondrogenesis

Early loss of up to 50% of cells is common for in vitro chondrogenesis of mesenchymal stromal cells (MSC) in pellet culture, reducing the efficacy and the tissue yield for cartilage engineering. Enhanced proliferation could compensate for this unwanted effect, but relevant signaling pathways remain largely unknown. The aim of this study was to identify the contribution of bone morphogenetic protein (BMP), fibroblast growth factor (FGF), insulin‐like growth factor (IGF), and hedgehog (HH) signaling toward cell proliferation during chondrogenesis and investigate whether a further mitogenic stimulation is possible and promising. Human MSC were subjected to chondrogenesis in the presence or absence of pathway inhibitors or activators up to Day 14 or from Days 14 to 28, before proliferation, DNA and proteoglycan content were quantified. [3H]‐thymidine incorporation revealed arrest of proliferation on Day 3, after which cell division was reinitiated. Although BMP signaling was essential for proliferation throughout chondrogenesis, IGF signaling was relevant only up to Day 14. In contrast, FGF and HH signaling drove proliferation only from Day 14 onward. Early BMP4, IGF‐1, or FGF18 treatment neither prevented early cell loss nor allowed further mitogenic stimulation. However, application of the HH‐agonist purmorphamine from Day 14 increased proliferation 1.44‐fold (p < 0.05) and late BMP4‐application enhanced the DNA and proteoglycan content, with significant effects on tissue yield. Conclusively, a differential and phase‐dependent contribution of the four pathways toward proliferation was uncovered and BMP4 treatment was promising to enhance tissue yield. Culture forms less prone to size limitations by nutrient/oxygen gradients and a focus on early apoptosis prevention may be considered as the next steps to further enhance chondrocyte formation from MSC.     

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.     

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.     

A Recombinant Human TGF-β1 Fusion Protein with Collagen-Binding Domain Promotes Migration, Growth, and Differentiation of Bone Marrow Mesenchymal Cells

A continuous source of osteoblasts for normal bone maintenance, as well as remodeling and regeneration during fracture repair, is ensured by the mesenchymal osteoprogenitor stem cells of the bone marrow (BM). The differentiation and maturation of osteoprogenitor cells into osteoblasts are thought to be modulated by transforming growth factors-β (TGF-β1 and TGF-β2) and TGF-β-related bone morphogenetic proteins (BMPs). To define the responses of mesenchymal osteoprogenitor stem cells to several growth factors (GFs), we cultured Fischer 344 rat BM cells in a collagen gel medium containing 0.5% fetal bovine serum for prolonged periods of time. Under these conditions, survival of BM mesenchymal stem cells was dependent on the addition of GFs. Recombinant hTGF-β1-F2, a fusion protein engineered to contain an auxiliary collagen binding domain, demonstrated the ability to support survival colony formation and growth of the surviving cells, whereas commercial hTGF-β1 did not. Initially, cells were selected from a whole BM cell population and captured inside a collagen network, on the basis of their survival response to added exogenous GFs. After the 10-day selection period, the surviving cells in the rhTGF-β1-F2 test groups proliferated rapidly in response to serum factors (10% FBS), and maximal DNA synthesis levels were observed. Upon the addition of osteoinductive factors, osteogenic differentiation in vitro was evaluated by the induction of alkaline phosphatase (ALP) expression, the production of osteocalcin (OC), and the formation of mineralized matrix. Concomitant with a down-regulation of cell proliferation, osteoinduction is marked by increased ALP expression and the formation of colonies that are competent for mineralization. During the induction period, when cells organize into nodules and mineralize, the expression of OC was significantly elevated along with the onset of extracellular matrix mineralization. Differentiation of BM mesenchymal stem cells into putative bone cells as shown by increased ALP, OC synthesis, and in vitro mineralization required the presence of specific GFs, as well as dexamethasone (dex) and β-glycerophosphate (β-GP). Although rhTGF-β1-F2-selected cells exhibited the capacity to mineralize, maximal ALP activity and OC synthesis were observed in the presence of rhBMPs. We further report that a novel rhTGF-β1-F2 fusion protein, containing a von Willebrand's factor-derived collagen binding domain combined with a type I collage matrix, is able to capture, amplify, and stimulate the differentiation of a population of cells present in rat BM. When these cells are subsequently implanted in inactivated demineralized bone matrix (iDBM) and/or diffusion chambers into older rats they are able to produce bone and cartilage. The population of progenitor cells captured by rhTGF-β1-F2 is distinct from the committed progenitor cells captured by rhBMPs, which exhibit a considerably more differentiated phenotype.     

A recombinant human TGF-beta1 fusion protein with collagen-binding domain promotes migration, growth, and differentiation of bone marrow mesenchymal cells.

A continuous source of osteoblasts for normal bone maintenance, as well as remodeling and regeneration during fracture repair, is ensured by the mesenchymal osteoprogenitor stem cells of the bone marrow (BM). The differentiation and maturation of osteoprogenitor cells into osteoblasts are thought to be modulated by transforming growth factors-beta (TGF-beta1 and TGF-beta2) and TGF-beta-related bone morphogenetic proteins (BMPs). To define the responses of mesenchymal osteoprogenitor stem cells to several growth factors (GFs), we cultured Fischer 344 rat BM cells in a collagen gel medium containing 0.5% fetal bovine serum for prolonged periods of time. Under these conditions, survival of BM mesenchymal stem cells was dependent on the addition of GFs. Recombinant hTGF-beta1-F2, a fusion protein engineered to contain an auxiliary collagen binding domain, demonstrated the ability to support survival colony formation and growth of the surviving cells, whereas commercial hTGF-beta1 did not. Initially, cells were selected from a whole BM cell population and captured inside a collagen network, on the basis of their survival response to added exogenous GFs. After the 10-day selection period, the surviving cells in the rhTGF-beta1-F2 test groups proliferated rapidly in response to serum factors (10% FBS), and maximal DNA synthesis levels were observed. Upon the addition of osteoinductive factors, osteogenic differentiation in vitro was evaluated by the induction of alkaline phosphatase (ALP) expression, the production of osteocalcin (OC), and the formation of mineralized matrix. Concomitant with a down-regulation of cell proliferation, osteoinduction is marked by increased ALP expression and the formation of colonies that are competent for mineralization. During the induction period, when cells organize into nodules and mineralize, the expression of OC was significantly elevated along with the onset of extracellular matrix mineralization. Differentiation of BM mesenchymal stem cells into putative bone cells as shown by increased ALP, OC synthesis, and in vitro mineralization required the presence of specific GFs, as well as dexamethasone (dex) and beta-glycerophosphate (beta-GP). Although rhTGF-beta1-F2-selected cells exhibited the capacity to mineralize, maximal ALP activity and OC synthesis were observed in the presence of rhBMPs. We further report that a novel rhTGF-beta1-F2 fusion protein, containing a von Willebrand's factor-derived collagen binding domain combined with a type I collage matrix, is able to capture, amplify, and stimulate the differentiation of a population of cells present in rat BM. When these cells are subsequently implanted in inactivated demineralized bone matrix (iDBM) and/or diffusion chambers into older rats they are able to produce bone and cartilage. The population of progenitor cells captured by rhTGF-beta1-F2 is distinct from the committed progenitor cells captured by rhBMPs, which exhibit a considerably more differentiated phenotype.     

Inhibition of cell proliferation and protease activity by cartilage factors and heparin

Proliferating rat smooth muscle cells and fibroblasts have membrane-associated protease activity. High concentrations of heparin inhibited membrane-associated protease activity and cell proliferation, while low concentration of heparin promoted smooth muscle cell proliferation. The inhibition of protease activity and proliferation was abolished when heparin was treated with protamine sulfate or when acid treated fetal calf serum was used. Heparin required the presence of an acid labile factor(s) in serum for the inhibition of protease activity and proliferation. Heparin and antithrombin III in the presence of acid-treated fetal calf serum did not inhibit cell proliferation or protease activity. Cartilage factors isolated from bovine nasal cartilage containing trypsin inhibitory activity, but not papain inhibitory activity, inhibited rat smooth muscle and fibroblast proliferation and surface associated protease activity. The cartilage factors did not require acid-labile components in the fetal calf serum for the inhibitory activity. The inhibitory activity due to heparin and cartilage factors was not permanent under our experimental condition. Protein synthesis was not inhibited by heparin or the cartilage factors. In rat smooth muscle cells and fibroblasts, the expression of surface-associated protease activity was related to the proliferative state of the cells. Surface protease activity was only present on proliferating cells. When surface protease activity was inhibited by high concentrations of heparin in the presence of an acid-labile serum component(s) or cartilage factors, cell proliferation was also inhibited.     

Collagen expression during teratocarcinoma cell line-induced endochondral bone tumor

Collagen immunotyping by indirect immunofluorescence was performed in order to investigate the sequential development of bone formation. Osseous tumors were obtained after subcutaneous injection of 3/A/1D-1 teratocarcinoma cell line into 129/Sv mice (Nicolas et al., 1980). Frozen sections of developing tumors were incubated with specific antibodies directed against Types I, II, III, IV, and IX collagens. On Day 9, the expression of Type I and Type III collagens was correlated with the proliferation of mesenchymal cells. From Day 10, chondrogenesis was characterized by the occurrence of cartilaginous collagens, Types II and IX, in the cartilage matrix. Type IV collagen was also detected in focal areas and revealed vascular invasion of the tumor. On Day 13, osteogenesis was demonstrated by the presence of Type I collagen in the bone matrix coating the surfaces. Immunolocalization of Type III collagen on the hemopoietic elements corresponded with the bone remodeling. The sequential transitions of collagen types confirm the development of an endochondral bone tumor. These results suggest that 3/A/1D-1 teratocarcinoma cell line constitutes a valuable system for in vitro study of endochondral bone formation and cell differentiation.     

Inhibition of induced endochondral bone development in caffeine-treated rats

We have addressed questions raised by the observation in fetal rats of delayed ossification induced by caffeine at maternal doses above 80 mg/kg body weight per day. The effect of caffeine on endochondral bone development and mineralization has been studied in an experimental model system of bone formation which involves implantation of demineralized bone particles (DBP) in subcutaneous pockets of young growing rats. Caffeine's effects on cellular events associated with endochondral ossification were examined directly by quantitating cellular mRNA levels of chondrocyte and osteoblast growth and differentiation markers in DBP implants from caffeine-treated rats harvested at specific stages of development (day 7 through day 15). Oral caffeine administration to rats implanted with DBP resulted in a dose dependent inhibition of the formation of cartilage tissue in the implants. Histologic examination of the implants revealed a decrease in the number of cells which were transformed to chondrocytes compared to control implants. Those cartilaginous areas that did form, however, proceeded through the normal sequelae of calcified cartilage and bone formation. At the 100 mg/kg dose, cellular levels of mRNA for histone, collagen type II, and TGFβ were all reduced by greater than 40% of control implants consistent with the histological findings. Alkaline phosphatase activity in the implants and mRNA levels for proteins reflecting the hypertrophic chondrocyte and bone phenotype, collagen type I and osteocalcin were markedly decreased compared to controls. Lower doses of 50 and 12.5 mg/kg caffeine also resulted in decreased cellular proliferation and transformation to cartilage histologically and reflected by significant inhibition of type II collagen mRNA levels (day 7). The effects of caffeine on gene expression observed in vivo during the period of bone formation (day 11 to day 15) in the DBP model were similar to the inhibited expression of H4, alkaline phosphatase, osteocalcin, and osteopontin found in fetal rat calvarial derived osteoblast cultures following 24 hour exposure of the cultures to 0.4 mM caffeine. Thus the observed delayed mineralization in the fetal skeleton associated with caffeine appears to be related to an inhibition of endochondral bone formation at the early stages of proliferation of undifferentiated mesenchymal cells to cartilage specific cells as well as at later stages of bone formation.     

Correlation of the appearance of γ-carboxyglutamic acid with the onset of mineralization in developing endochondral bone

γ-Carboxyglutamic acid (Gla) is a constituent of the non-collagenous bone protein osteocalcin. The appearance of γ-carboxyglutamic acid during $\text{de}\text{novo}$ differentiation and development of endochondral bone has been correlated with the onset of mineralization. Discrete stages of endochondral bone development were studied by subcutaneous implantation of demineralized rat diaphyseal bone matrix. Residual Gla in acid-demineralized bone matrix was lost rapidly on implantation. Gla levels were basal during mesenchymal cell proliferation (day 3) and chondrogenesis (days 5–7). Gla and calcium levels began to increase during cartilage mineralization (day 9) and continuously increased after day 10 concomitant with bone differentiation.     

Cartilage-specific RBPjκ-dependent and -independent Notch signals regulate cartilage and bone development

The Notch signaling pathway has emerged as an important regulator of endochondral bone formation. Although recent studies have examined the role of Notch in mesenchymal and chondro-osteo progenitor cell populations, there has yet to be a true examination of Notch signaling specifically within developing and committed chondrocytes, or a determination of whether cartilage and bone formation are regulated via RBPjκ-dependent or -independent Notch signaling mechanisms. To develop a complete understanding of Notch signaling during cartilage and bone development we generated and compared general Notch gain-of-function (Rosa-NICD(f/+)), RBPjκ-deficient (Rbpjκ(f/f)), and RBPjκ-deficient Notch gain-of-function (Rosa-NICD(f/+);Rbpjκ(f/f)) conditional mutant mice, where activation or deletion of floxed alleles were specifically targeted to mesenchymal progenitors (Prx1Cre) or committed chondrocytes (inducible Col2Cre(ERT2)). These data demonstrate, for the first time, that Notch regulation of chondrocyte maturation is solely mediated via the RBPjκ-dependent pathway, and that the perichodrium or osteogenic lineage probably influences chondrocyte terminal maturation and turnover of the cartilage matrix. Our study further identifies the cartilage-specific RBPjκ-independent pathway as crucial for the proper regulation of chondrocyte proliferation, survival and columnar chondrocyte organization. Unexpectedly, the RBPjκ-independent Notch pathway was also identified as an important long-range cell non-autonomous regulator of perichondral bone formation and an important cartilage-derived signal required for coordinating chondrocyte and osteoblast differentiation during endochondral bone development. Finally, cartilage-specific RBPjκ-independent Notch signaling likely regulates Ihh responsiveness during cartilage and bone development.     

Membrane-type MMPs enable extracellular matrix permissiveness and mesenchymal cell proliferation during embryogenesis

Peri-cellular remodeling of mesenchymal extracellular matrices is considered a prerequisite for cell proliferation, motility and development. Here we demonstrate that membrane-type 3 MMP, MT3-MMP, is expressed in mesenchymal tissues of the skeleton and in peri-skeletal soft connective tissue. Consistent with this localization, MT3-MMP-deficient mice display growth inhibition tied to a decreased viability of mesenchymal cells in skeletal tissues. We document that MT3-MMP works as a major collagenolytic enzyme, enabling cartilage and bone cells to cleave high-density fibrillar collagen and modulate their resident matrix to make it permissive for proliferation and migration. Collectively, these data uncover a novel extracellular matrix remodeling mechanism required for proper function of mesenchymal cells. The physiological significance of MT3-MMP is highlighted in mice double deficient for MT1-MMP and MT3-MMP. Double deficiency transcends the combined effects of the individual single deficiencies and leads to severe embryonic defects in palatogenesis and bone formation incompatible with life. These defects are directly tied to loss of indispensable collagenolytic activities required in collagen-rich mesenchymal tissues for extracellular matrix remodeling and cell proliferation during embryogenesis.     

Endothelin-1 expression in long-term cultures of fetal rat calvarial osteoblasts: regulation by BMP-7

Endothelin-1 (ET-1) is a vasoactive peptide that modulates bone metabolism via regulatory effects on osteoblasts, chondrocytes, and osteoclasts. While ET-1 may circulate in the blood stream, tissue-specific expression of this peptide is more physiologically relevant. In the present study we measured ET-1 synthesis in sections of fetal rat calvaria (FRC) and in cultured FRC osteoblasts. Regulation of ET-1 synthesis in FRC osteoblasts by bone morphogenetic protein-7 (BMP-7) and transforming growth factor-beta1 (TGF-beta1) also was examined. Immunohistochemical analysis revealed ET-1 staining in calvarial osteoblasts, endothelial cells, and osteocytes. ET-1 mRNA expression was detected in cultured FRC cells and ET-1 peptide was present in conditioned media. During long-term culture of FRC cells (26 days) ET-1 peptide production rose sharply and peaked during the time of cellular proliferation (Days 0-3) then returned to baseline levels by Day 18, when mineralized nodules were forming. Treatment of FRC cells with BMP-7 enhanced ET-1 levels by three-fold on Day 3 and enhanced nodule formation by 15-fold on Day 26. To determine whether ET-1 was involved in an autocrine manner in BMP-7-induced nodule formation, cells were cultured in the presence of BMP-7 and BQ-123, an ET(A) receptor antagonist. BQ-123 had no effect on nodule formation in control or BMP-7-treated cells, indicating that osteoblast-derived ET-1 regulates other cell types in vivo during the bone formation process.     

Parallels between development of embryonic and matrix-induced endochondral bone

Endochondral bone formation can take place in the embryo, during fracture healing, or in postnatal animals after induction by implanted demineralized bone matrix. This matrix-induced bone formation recapitulates the embryonic sequence of bone formation morphologically and biochemically. The steps in bone formation in both systems include differentiation of cartilage from mesenchyme, cartilage maturation, invasion of the cartilage by blood vessels and marrow precursors, and formation of bone and bone marrow. Recently, bone inductive molecules from demineralized bone matrix have been purified, sequenced and produced as recombinant proteins. While there are similarities between bone development in the embryo and that after induction by these purified molecules, the molecules responsible for bone induction in the embryo have not yet been defined. Because of similarities between the two methods of bone formation, studies of bone induction by demineralized bone matrix may help to elucidate mechanisms of embryonic bone induction.