Growth behaviour of human mononuclear cells derived from bone marrow and cord blood on a collagen carrier for osteogenic regeneration]

We investigated the biocompatibility and osteogenetic potency of a porcine collagen I/III carrier in a human bone marrow and cord blood cell culture system. METHODS: Human mesenchymal mononuclear cells were isolated from cord blood and iliac crest bone marrow and cultivated in various cell densities on a semipermeable porcine collagen I/III carrier. After 14 days of in vitro cultivation both cultures were subjected to osteogenic stimulation by dexamethasone, ascorbic acid and beta-glycerol phosphate (DAG) until day 40. Semiquantitative immunochemical evaluation based on osteoblastic and progenitor cell markers was then done. RESULTS: With regard to the minimal local cell density required for growth and osteogenic differentiation, cord blood derived progenitor cells showed lower tolerance in comparison with bone marrow derived cells. For both cell culture systems three-dimensional growth and calcification within the collagen fibres were seen after osteogenic stimulation. CONCLUSION: Human cord blood and bone marrow derived mesenchymal stem cell are capable of differentiating into osteoblasts after incubation with a collagen I/III biomaterial.     

Development of osteogenic tissue in diffusion chambers from early precursor cells in bone marrow of adult rats

Diffusion chambers containing bone marrow cells from adult rats were implanted intraperitoneally into rat hosts and cultured in vivo for up to 64 days. Biochemical and histological analyses of the contents of the chambers demonstrate that a connective tissue consisting of bone, cartilage and fibrous tissues is formed by precursor cells present in marrow stroma. The amounts of osteogenic tissue and DNA are directly correlated with time of implantation and with number of cells inoculated. In the chambers there is initial formation of fibrous tissue which is strongly reactive to collagen type III, laminin and fibronectin. In areas of osteogenesis which appear later within this fibrous anlage, expression of collagen type III, laminin and fibronectin decrease and collagen types I and II increase in association with bone and cartilage respectively. Where osteogenesis does not develop, fibrous tissue continues to express collagen type III. The sequential expression of the different extracellular matrix components is similar to that previously observed during osteogenic differentiation in embryonic and adult developmental systems. It is concluded that the formation of fibrous and osteogenic tissues in diffusion chambers by precursor cells present in adult marrow, resembles the normal developmental process.     

Effect of uniaxial stretching on rat bone mesenchymal stem cell: orientation and expressions of collagen types I and III and tenascin-C

Bone marrow mesenchymal stem cells (BMSC) have the potential to differentiate into a variety of cell types like osteoblasts, chondroblasts, adipocytes, etc. It is well known that mechanical forces regulate the biological function of cells. The aim of this study was to investigate the effect of uniaxial stretching on the orientation and biological functions of BMSC. Rat BMSCs were harvested from femoral and tibial bone marrow by density gradient centrifugation. Cells from passages 1-6 were characterized by flow cytometry using monoclonal antibodies. The recovered cells were stably positive for the markers CD90 and CD44 and negative for CD34 and CD45. A cyclic 10% uniaxial stretching at 1Hz was applied on rat BMSC for different time-courses. The length, width, and orientation of the cells were subsequently determined. Expression of collagen types I and III and tenascin-C mRNAs was measured by real-time RT-PCR, and the synthesis of these receptors was determined by radioimmunoassay. Results showed that uniaxial stretching lengthened and rearranged the cells. Compared with control groups, expression of collagen types I and III mRNAs was up-regulated after 12-h of stretching, while significant increase in synthesis of the two collagen protein types was not observed until after 24-h stretching. The expression of tenascin-C mRNA was significantly increased after a 24-h stretching. These data suggest that cyclic stretching promotes the synthesis of collagen types I and III and tenascin-C by the rat BMSC.     

Collagen synthesis by murine bone marrow cell culture

Collagen types synthesized by murine bone marrow cells were studied and the effect of lithium chloride on collagen biosynthesis in vitro was investigated. In the liquid culture system used, an adherent, mixed cell population supports hemopoiesis. Radioactive labeling of cell cultures and subsequent fractionation with ammonium sulfate, enzyme digestion, immune precipitation, and gel electrophoresis indicated that the bone marrow cells synthesized precursors to collagen types I, III, and IV, and fibronectin. A previously undescribed molecule or fragment with an apparent molecular weight of 17,000 daltons that was susceptible to bacterial collagenase and containing no interchain disulfide bonds was also identified in the culture media of both control and lithium-treated cells. Lithium treatment did not affect the types of collagen synthesized, although the relative proportions of collagen types may differ from controls. However, lithium does have an effect on the appearance of some, as yet unidentified, non-collagenous components in the cell culture media.     

The expression of the fibrillar collagen genes during fracture healing: heterogeneity of the matrices and differentiation of the osteoprogenitor cells

The cells that express the genes for the fibrillar collagens, types I, II, III and V, during callus development in rabbit tibial fractures healing under stable and unstable mechanical conditions were localized. The fibroblast-like cells in the initial fibrous matrix express types I, III and V collagen mRNAs. Osteoblasts, and osteocytes in the newly formed membranous bone under the periosteum, express the mRNAs for types I, III and V collagens, but osteocytes in the mature trabeculae express none of these mRNAs. Cartilage formation starts at 7 days in calluses forming under unstable mechanical conditions. The differentiating chondrocytes express both types I and II collagen mRNAs, but later they cease expression of type I collagen mRNA. Both types I and II collagens were located in the cartilaginous areas. The hypertrophic chondrocytes express neither type I, nor type II, collagen mRNA. Osteocalcin protein was located in the bone and in some cartilaginous regions. At 21 days, irrespective of the mechanical conditions, the callus consists of a layer of bone; only a few osteoblasts lining the cavities now express type I collagen mRNA.We suggest that osteoprogenitor cells in the periosteal tissue can differentiate into either osteoblasts or chondrocytes and that some cells may exhibit an intermediate phenotype between osteoblasts and chondrocytes for a short period. The finding that hypertrophic chondrocytes do not express type I collagen mRNA suggests that they do not transdifferentiate into osteoblasts during endochondral ossification in fracture callus.     

Characterization of reticulofibroblastoid colonies (CFU-RF) derived from bone marrow and long-term marrow culture monolayers

The maintenance of hemopoietic precursors in long-term liquid bone marrow cultures (LTBMC) is associated with the presence of an adherent stromal layer composed of heterogeneous cell populations. We have used a culture assay to promote the growth of one of its cellular components and characterize its properties. Freshly obtained bone marrow cells and cells derived from the adherent layer of LTBMC were grown in methylcellulose-clotted plasma in the presence of phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM), hydrocortisone (HC), and citrated normal human plasma. Both sources contained cells (CFU-RF) that gave rise to colonies of cells with a reticulofibroblastoid appearance. In the presence of HC, most colonies contained lipid-laden cells. Colonies could be further propagated as adherent layers when transferred into liquid cultures. These cells produced laminin, fibronectin, and collagen types I, III, IV, and V. They were negative for Von Willebrand factor VIII. The ability to synthesize laminin and collagen type IV distinguished these cells from a population of previously described bone marrow fibroblasts (CFU-F). The relationship of CFU-RF to hemopoietic precursors was investigated using patients with chronic myeloid leukemia and bone marrow transplant recipients. Cells within CFU-RF-derived colonies were uniformly negative for the Philadelphia chromosome, thus making it unlikely that they belonged to the malignant hemopoietic clone. CFU-RF-derived colonies in bone marrow transplant recipients were found to be exclusively of host origin. Both observations support the view that CFU-RF is not part of the repertoire of hemopoietic stem cells.     

Type I collagen-induced osteoblastic differentiation of bone-marrow cells mediated by collagen-alpha2beta1 integrin interaction

Bone marrow cells are multipotent cells. When bone marrow cells were cultured with type I collagen matrix gels, they showed high alkaline phosphatase activity, collagen synthesis, and formed mineralized tissues. Furthermore, cells expressed osteocalcin and bone sialoprotein genes, which are osteoblast-specific genes. These findings indicate that type I collagen matrix gels induce osteoblastic differentiation of bone marrow cells. Type I collagen interacts with the alpha 2 beta 1 integrin receptor on the cell membrane and mediates extracellular signals into cells. DGEA peptide is a cell-binding domain of type I collagen molecule. When collagen-integrin interaction was interrupted by the addition of Asp-Gly-Glu-Ala (DGEA) peptide to the culture, the expression of osteoblastic phenotypes of bone marrow cells was inhibited. Furthermore, anti-alpha 2 integrin antibody, which interacts with alpha subunit of integrin and blocks the binding of integrin with collagen, suppressed the expression of osteoblastic phenotypes. These findings imply that collagen-alpha 2 beta 1 integrin interaction is an important signal for the osteoblastic differentiation of bone marrow cells.     

Immunohistochemical distribution of collagens types IV, V, and VI and of pro-collagens types I and III in human alveolar bone and dentine

The aim of the present study was to characterize the composition of the organic matrix in alveolar jaw bone and dentine using antibodies against pro-collagens Types I and III and collagens Types IV, V, and VI. After demineralization of oral hard tissues in 0.2 N HCl, antigenicity was well preserved and the distribution of the pro-collagens and collagens could be demonstrated. Staining for pro-collagen Type I was prominent around osteoblasts and in pre-dentine, indicating active de novo synthesis of Type I pro-collagen. Pro-collagen Type I was ubiquitous but was less abundant in bone and dentine, whereas pro-collagen Type III was seen only in areas of bone remodeling, in peritubular spaces, and in pre-dentine. Type IV collagen was limited to the basement membranes of vessels in osteons and bone marrow. Type V collagen was detected neither in pre-dentine nor in bone. In contrast, Type VI collagen was found in dentine and bone, showing a faint but homogeneous staining which, similarly to pro-collagen Type III, was pronounced around osteoblasts and in pre-dentine, areas of active bone and dentine formation. This study showed that the organic matrix of dentine and bone contains Type VI as well as Type I collagen. Pro-collagen Type III (and to a lesser extent collagen Type VI) is transiently produced during new formation and remodeling of oral hard tissues, and disappears once the matrix calcifies. Type I pro-collagen qualifies as a general marker protein for increased osteoblastic activity. We conclude that immunostaining for the different collagen/pro-collagen types can be used to assess normal or abnormal stages of bone/dentine formation.     

Strain-related collagen gene expression in human osteoblast-like cells

The gene expression of cells in the musculoskeletal system, such as in bone, cartilage, ligament and tendon, is profoundly affected by mechanical loading. Previous studies have demonstrated that the expression of many genes, including collagen types I and III, are affected by mechanical strain in diverse cell types, such as human osteoblast-like SaOs-2 cells. However, whether the effect of mechanical loading on collagen gene expression is strain-related remains unclear. The goal of this study was to determine the relationship between mechanical strain and the gene expression of collagen types I and III in SaOs-2 cells. A Flexercell cellular mechanical loading system was used to subject SaOs-2 cells to equibiaxial cyclic tensile stress at a rate of 0.5 Hz with various strains of 5%, 7.5%, 10%, and 12.5% for 24 h. The relative amount of mRNA of both collagen I and collagen III increased at 5% strain compared with that of the control. As the strain increased, the relative amount of mRNA of collagen I remained stable at strain levels up to 12.5%. However, the mRNA for collagen III began to drop when the strain was greater than 5%, until a 10% strain was reached. From the application of a 10% strain through the maximum loading of a 12.5% strain, the relative amount of collagen III mRNA remained stable at amounts lower than that of the control. Thus, the gene expression of collagen types I and III responds differentially to mechanical strain at various magnitudes.     

Activity of matrix metalloproteinase-9 against native collagen types I and III

Interstitial collagen types I, II and III are highly resistant to proteolytic attack, due to their triple helical structure, but can be cleaved by matrix metalloproteinase (MMP) collagenases at a specific site, approximately three-quarters of the length from the N-terminus of each chain. MMP-2 and -9 are closely related at the structural level, but MMP-2, and not MMP-9, has been previously described as a collagenase. This report investigates the ability of purified recombinant human MMP-9 produced in insect cells to degrade native collagen types I and III. Purified MMP-9 was able to cleave the soluble, monomeric forms of native collagen types I and III at 37 degrees C and 25 degrees C, respectively. Activity against collagens I and III was abolished by metalloproteinase inhibitors and was not present in the concentrated crude medium of mock-transfected cells, demonstrating that it was MMP-9-derived. Mutated, collagenase-resistant type I collagen was not digested by MMP-9, indicating that the three-quarters/one-quarter locus was the site of initial attack. Digestion of type III collagen generated a three-quarter fragment, as shown by comparison with MMP-1-mediated cleavage. These data demonstrate that MMP-9, like MMP-2, is able to cleave collagens I and III in their native form and in a manner that is characteristic of the unique collagenolytic activity of MMP collagenases.     

Enhanced expression of TGF-beta and c-fos mRNAs in the growth plates of developing human long bones

The expression of mRNAs for type I and type II procollagens, transforming growth factor-beta (TGF-beta) and c-fos was studied in developing human long bones by Northern blotting and in situ hybridization. The cells producing bone and cartilage matrix were identified by hybridizations using cDNA probes for types I and II collagen, respectively. Northern blotting revealed that the highest levels of TGF-beta mRNA were associated with the growth plates. By in situ hybridization, this mRNA was localized predominantly in the osteoblasts and osteoclasts of the developing bone, in periosteal fibroblasts and in individual bone marrow cells. These findings are consistent with the view that TGF-beta may have a role in stimulation of type I collagen production and bone formation. Only a low level of TGF-beta mRNA was detected in cartilage where type II collagen mRNA is abundant. In Northern hybridization, the highest levels of c-fos mRNA were detected in epiphyseal cartilage. In situ hybridization revealed two cell types with high levels of c-fos expression: the chondrocytes bordering the joint space and the osteoclasts of developing bone. These differential expression patterns suggest specific roles for TGF-beta and c-fos in osseochondral development.     

Transfer of proalpha2(I) cDNA into cells of a murine model of human Osteogenesis Imperfecta restores synthesis of type I collagen comprised of alpha1(I) and alpha2(I) heterotrimers in vitro and in vivo

The oim mouse is a model of human Osteogenesis Imperfecta (OI) that has deficient synthesis of proalpha2(I) chains. Cells isolated from oim mice synthesize alpha1(I) collagen homotrimers that accumulate in tissues. To explore the feasibility of gene therapy for OI, a murine proalpha2(I) cDNA was inserted into an adenovirus vector and transferred into bone marrow stromal cells isolated from oim mice femurs. The murine cDNA under the control of the cytomegalovirus early promoter was expressed by the transduced cells. Analysis of the collagens synthesized by the transduced cells demonstrated that the cells synthesized stable type I collagen comprised of alpha1(I) and alpha2(I) heterotrimers in the correct ratio of 2:1. The collagen was efficiently secreted and also the cells retained the osteogenic potential as indicated by the expression of alkaline phosphatase activity when the transduced cells were treated with recombinant human bone morphogenetic protein 2. Injection of the virus carrying the murine proalpha2(I) cDNA into oim skin demonstrated synthesis of type I collagen comprised of alpha1 and alpha2 chains at the injection site. These preliminary data demonstrate that collagen genes can be transferred into bone marrow stromal cells as well as fibroblasts in vivo and that the genes are efficiently expressed. These data encourage further studies in gene replacement for some forms of OI and use of bone marrow stromal cells as vehicles to deliver therapeutic genes to bone.     

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.     

Osteoblast-related gene expression of bone marrow cells during the osteoblastic differentiation induced by type I collagen

Bone marrow contains multipotent cells that differentiate into fibroblasts, adipocytes, and osteoblasts. Recently we found that type I collagen matrix induced the osteoblastic differentiation of bone marrow cells. Three weeks after cells were cultured with type I collagen, they formed mineralized tissues. In this study, we investigated the expression of osteoblast-related genes (alkaline phosphatase, osteocalcin, bone sialoprotein, osteopontin, and cbfa-1) during the osteoblastic differentiation. The expression of alkaline phosphatase and osteopontin genes increased time-dependently during the osteoblastic differentiation. Osteocalcin and bone sialoprotein genes were expressed in cells that formed mineralized tissues, and both were expressed only after cells reached the mineralized tissue-formation stage. On the other hand, the cbfa-1 gene was expressed from the early differentiation stage. The Asp-Gly-Glu-Ala (DGEA) amino acid domain of type I collagen interacts with the alpha2beta1 integrin receptor on the cell membrane and mediates extracellular signals into cells. When the collagen-integrin interaction was interrupted by the addition of DGEA peptide to the culture, the expression of osteoblastic phenotypes of bone marrow cells was inhibited. These findings imply that the collagen-alpha2beta1 integrin interaction is an important signal for the osteoblastic differentiation of bone marrow cells.     

Localization of the expression of types I, III, and IV collagen, TGF-beta 1 and c-fos genes in developing human calvarial bones

Total RNA extracted from developing calvarial bones of 15- to 18-week human fetuses was studied by Northern hybridization: in addition to high levels of type I collagen mRNAs, the presence of mRNAs for type III and type IV collagen, TGF-beta and c-fos was observed. In situ hybridization of sections containing calvarial bone, overlying connective tissues, and skin was employed to identify the cells containing these mRNAs. Considerable variation was observed in the distribution of pro alpha 1(I) collagen mRNA in osteoblasts: the amount of the mRNA in cells at or near the upper surface of calvarial bone was distinctly greater than that in cells at the lower surface, indicating the direction of bone growth. High levels of type I collagen mRNAs were also detected in fibroblasts of periosteum, dura mater, and skin. Type III collagen mRNA revealed a considerably different distribution: the highest levels were detected in upper dermis, lower levels were seen in fibroblasts of the periosteum and the fibrous mesenchyme between bone spiculas, and none was seen in osteoblasts. Type IV collagen mRNAs were only observed in the endothelial cells of blood capillaries. Immunohistochemical localization of type III and IV collagens agreed well with these observations. The distribution of TGF-beta mRNA resembled that of type I collagen mRNA. In addition, high levels of TGF-beta mRNA were observed in osteoclasts of the calvarial bone. These cells, responsible for bone resorption, were also found to contain high levels of c-fos mRNA. Production of TGF-beta by osteoclasts and its activation by the acidic environment could form a link between bone resorption and new matrix formation.     

Differential expression of fibrillar collagen genes during callus formation

An experimental fracture healing model in the rat tibio-fibular bone was employed to study the appearance of messenger RNAs for types I, II and III collagens during endochondral fracture repair. Total RNA was extracted from normal bone and from callus tissue at various time points. The total RNAs were analyzed in Northern hybridization for their contents of procollagen mRNAs using specific cDNA clones. The results show that during the first week of fracture repair type III collagen mRNA is increased to the greatest extent, followed by type II collagen mRNA during the second week. The 28-day callus resembles bone by containing mainly type I collagen mRNAs and very little type II or III collagen mRNA.     

The effects of mechanical stability on the macromolecules of the connective tissue matrices produced during fracture healing. I. The collagens

Summary The distribution of types I, II, III, V and IX collagens in healing fractures of the rabbit tibia has been demonstrated by immunofluorescent techniques. It has also been shown that the mechanical stability of the healing fracture affects both the distribution and types of the collagens present.The initial fibrous matrix contains types III and V collagens; type I collagen was only located in this matrix if unfixed tissue was used. In mechanically stable fractures, cancellous bone forms over the entire periosteal surface by 5–7 days; type I collagen is laid down within the previous fibrous matrix. The trabeculae are heterogeneous in their collagen content. The cavities contain a matrix of types III and V collagens. Small nodules of cartilage may be present between 7 and 14 days; these contain types II and IX collagens.In mechanically unstable fractures, cancellous bone is initially formed away from the fracture gap. The fibrous tissue over the gap is replaced by cartilage; types II and IX collagens are laid down on the pre-existing fibrous matrix. The cartilage is replaced by endochondral ossification. At the ossification front, type I collagen is found around the chondrocyte lacunae of the spicules of cartilage. The new trabeculae contain a core of cartilage which is surrounded by a bone matrix of types I and V collagens.The fracture gaps are invaded by fibrous tissue, which contain types III and V collagens. This is later replaced by cancellous bone.     

Mechanisms underlying catabolic and anabolic functions of parathyroid hormone on bone by combination of culture systems of mouse cells

Since bone resorption and formation by continuous and intermittent parathyroid hormone (PTH) treatments involve various types of cells in bone, this study examined the underlying mechanism by combining culture systems using mouse primary calvarial osteoblasts and bone marrow cells. The PTH/PTHrP receptor (PTH1R) expression and the cAMP accumulation in response to PTH were increased in accordance with the differentiation of osteoblasts. Osteoclast formation was strongly induced by continuous PTH treatment in the monolayer co‐culture of osteoblasts and bone marrow cells, which was associated with RANKL expression in differentiated osteoblasts. Bone formation determined by ALP activity and the type I collagen mRNA expression was stimulated by intermittent PTH treatment in the monolayer co‐culture and in the bone marrow cell layer of the separated co‐culture in a double chamber dish, but not in the culture of bone marrow cells alone. The stimulation in the separated co‐culture, accompanied by IGF‐I production by osteoblasts, was abolished when bone marrow cells were derived from knockout mice of insulin‐receptor substrate‐1 (IRS‐1?/?) or when osteoblasts were from PTH1R?/? mice. We conclude that differentiated osteoblasts are most likely the direct target of both continuous and intermittent PTH, while bone marrow cells are likely the effector cells. The osteoblasts stimulated by continuous PTH express RANKL which causes osteoclastogenesis from the precursors in bone marrow via cell‐to‐cell contact, leading to bone resorption; while the osteoblasts stimulated by intermittent PTH secrete IGF‐I which activates IRS‐1 in osteoblast precursors in bone marrow via a paracrine mechanism, leading to bone formation. J. Cell. Biochem. 109: 755–763, 2010. © 2010 Wiley‐Liss, Inc.     

In vitro expression of osteoblastic markers in cells isolated from normal fetal and postnatal human bone and from bone of patients with osteogenesis imperfecta

We studied the expression of osteoblastic markers in cultured cells isolated from the bone of 15 patients with different clinical forms of osteogenesis imperfecta (OI) and of seven fetal and postnatal controls. Cultured bone cells of ten OI patients produced abnormal collagen type I. Similar to controls, OI bone cells produced predominantly collagen type I with traces of collagen types III and V. The 1,25(OH)₂ vitamin D₃-stimulated synthesis of osteocalcin, a specific osteoblastic marker protein, was similar in OI bone cells and age-matched controls. Bone cells from fetal controls and from patients with the perinatal lethal OI type II produced less osteocalcin than bone cells from postnatal controls and surviving OI patients. OI bone cells responded to parath.yroid hormone (PTH) by increased production of cAMP similar to controls. Bone cells from fetal controls and from OI type II donors showed a decreased response to PTH. Activity of the bone-liver-kidney isoenzyme alkaline phosphatase (AP) was detected in all control and OI bone cells. The expression of all osteoblastic markers was similar in bone cells producing abnormal collagen type I. These observations show that OI bone cells in vitro express a pattern of osteoblastic markers similar to age-matched control bone cells indicating that osteoblastic differentiation is not altered by the underlying defects of collagen type I metabolism in OI bone cells. © 1993 Wiley-Liss, Inc.