Partial isolation and characterization of a chemotactic factor from adult bovine bone for mesenchymal cells

Demineralized adult bone matrix has the capacity to initiate de novo ectopic endochondral bone formation 2-3 weeks following intramuscular implantation into suitable hosts. An early step in this process is the migration of mesenchymal cells to the implant site; these cells later differentiate into cartilage and bone. Adult bone has been shown to contain a number of bioactive factors, such as chemotactic factors for various cell types, including osteoblasts. We have used embryonic chick limb bud mesenchymal cells to construct an in vitro assay for testing chemotactic activity derived from bone matrix extracts. With a modified Boyden chamber, water-soluble components from a 4 M guanidinium chloride extract of demineralized adult bovine bone matrix were found to stimulate the directional migration of these chick embryonic limb bud mesenchymal cells as well as embryonic muscle-derived fibroblasts and cells derived from embryonic skin. The chemotactic activity was destroyed by treatment with heat (100 degrees C) or trypsin. Partial purification by molecular sieve chromatography suggested that the chemotactic factor(s) has a molecular weight of between 50,000 and 90,000. This factor can be separated from bone matrix-derived chondrogenic stimulating activity by either ion exchange or molecular sieve chromatography. These observations confirm that bone matrix contains a chemoattractant for mesenchymal cells that may be important for in vivo recruitment of cells as part of the process of ectopic bone formation or in cases of bone repair.     

Chemotaxis of muscle-derived mesenchymal cells to bone-inductive proteins of rat

Summary In this investigation we examined the chemotaxis of muscle-derived mesenchymal cells from neonatal rats to partially purified extracts of demineralized bone matrix with osteoinductive properties. Using the modified Boyden chamber assay and muscle-derived mesenchymal cells obtained from neonatal Long-Evans rats, we tested the chemotactic properties of the 4 M guanidine-HCl extract from demineralized rat bone matrix and fractions thereof that were derived from sequential Sepharose CL-6B, TSK-3000 and HPLC-C₁₈ chromatography. We have identified that those fractions exhibiting chemotactic properties were also osteoinductive. Therefore, demineralized bone matrix serves as its own soluble signal and insoluble substratum in the inductive process leading to endochondral bone formationin vivo.     

Chemotaxis of rat peritoneal macrophages induced by rat bone powder

Mononuclear phagocytes are frequently found adjacent to active bone-resorbing surfaces in both physiological and pathological bone resorption, being implicated as important cellular elements in the process of bone resorption. In this study an attempt was made to prepare several groups of peritoneal exudate cells (PEC) induced by bone powder (B-PEC), glycogen (G-PEC) or paraffin oil (P-PEC), to investigate the chemotactic response to bone powder suspension or bone matrix and the superoxide production. B-PEC migrated to bone powder or bone matrix more readily than the other exudate cells (G-PEC and P-PEC) did. An checkerboard analysis showed that there was a chemokinetic activity in bone matrix, besides chemotactic activity. Superoxide production by bone matrix, however, remained unchanged in each cell group. These results suggest that there is a macrophage subset possessing a specific migratory characteristic toward the bone tissue.     

Identification of a bone matrix-derived chemotactic factor

When demineralized bone matrix powder is implanted subcutaneously in the rat, the early responses involve the appearance and proliferation of mesenchymal cells at the site of implantation, followed by cartilage and bone formation. The ability of cells to migrate to the implant suggests that chemotaxis may be a critical event in this process. Therefore, using the modified Boyden chamber assay, we tested extracts of demineralized bone matrix for chemotactic activity. We have identified and partially purified, on molecular sieve chromatography, a heat labile and trypsin-sensitive protein (Mr = 60,000-70,000) that is a potent chemoattractant for mouse calvaria, osteoblast-like cells (MMB-1), but not for monocytes (putative osteoclast precursors). These findings suggest that chemotactic protein(s) have a significant role in the recruitment of osteoprogenitor cells to a site of bone repair.     

Chemotactic activity of theγ-carboxyglutamic acid containing protein in bone

Summary We have found that theγ-carboxyglutamic acid (GLA)-containing protein from bone (BGP, osteocalcin) has chemotactic activityin vitro for a number of cells which are found adjacent to endosteal bone surfacesin vivo. Using the Boyden chamber technique for measuring cell chemotaxisin vitro, we have shown that BGP is chemotactic for cultured human breast cancer cells, human and mouse monocytes, and for cultured rat osteosarcoma cells which have the characteristics of osteoblasts. The migration of these cells in response to BGP is unidirectional and not due to spontaneous or random migration. A synthetic peptide (Phe-Tyr-Gly-Pro-Val), which is identical to the carboxyterminal peptide cleaved from BGP when digested by trypsin, is also chemotactic for the same cells. BGP retains its chemotactic activity after conversion of theγ-carboxyglutamic acid residues to glutamic acid, indicating that this biological effect requires neitherγ-carboxyglutamate nor the ability of BGP to bind calcium. Since BGP is released from bone during states of increased bone turnover, it is possible that this chemotactic effect of the protein may be a mechanism for recruitment of these cells to sites of active bone remodeling.     

Unidirectional migration of osteosarcoma cells with osteoblast characteristics in response to products of bone resorption

Summary To investigate the mechanisms by which bone-forming cells are attracted to areas of bone resorption during bone remodeling, we have usedin vitro methods to look for signals released by resorbing bone, which may be chemotactic for cultured bone cells. We have found that cultured rat osteosarcoma cells, which have characteristics associated with the osteoblastic phenotype, migrate in a unidirectional manner in response to a signal released by resorbing bones. These cells also migrated unidirectionally in response to Type I collagen, which comprises 95% of the bone matrix. This phenomenon of chemotaxis of bone-forming cells to sites of previous resorption may be an important component of the process of bone remodeling and the coupling of bone formation to bone resorption.     

Chemotactic response of osteoblastlike cells to transforming growth factor beta

Transforming growth factor beta (TGF-beta) has multiple effects on bone cell metabolism in vitro but its exact role in bone remodeling still needs to be defined. Here we demonstrate that TGF-beta is chemotactic for osteoblastlike cells from fetal rat calvariae and osteoblastlike ROS 17/2.8 osteosarcoma cells. Maximal chemotaxis occurred at 5-15 pg/ml of TGF-beta and was observed with TGF-beta 1 and TGF-beta 2 at equivalent concentrations. Conditioned medium from osteoblastlike cells containing latent TGF-beta failed to stimulate chemotactic migration. However, chemotactic activity was observed in conditioned medium that had been transiently acidified. Since acidification is known to activate TGF-beta, these results suggest that only active TGF-beta is capable of inducing a chemotactic response. Preincubation of osteoblastlike cells with TGF-beta in concentrations from 10 pg/ml to 1 ng/ml for 48 h abolished a subsequent chemotactic response of these cells to TGF-beta, indicating that TGF-beta-induced chemotaxis is a transient phenomenon. Since TGF-beta may be released from the bone matrix and/or activated during bone resorption, the chemotactic activity of TGF-beta for osteoblastlike cells may be important for the recruitment of osteoblastlike cells to sites of bone remodeling.     

Chemotactic response of mesenchymal cells, fibroblasts and osteoblast-like cells to bone Gla protein

Bone gla protein (BGP) and decarboxylated bone gla protein (dBGP) were tested for chemotactic activity against stage 24 chick limb bud mesenchymal cells, chick embryonic muscle-derived fibroblasts, murine Balb/C 3T3 cells, and two lines of rat osteosarcoma cells, ROS-17/2.8 and -25/1. Both BGP and dBGP were potent chemoattractants for all the cell types except 3T3 cells. The dose response curves were bell-shaped, with maximal chemotactic response ranging from 5 pg/ml for ROS 25/1 cells to 10 ng/ml for the stage 24 limb bud cells. dBGP was equally potent a chemoattractant as BGP for all cell types tested indicating that the gamma-carboxylation of the glutamic acid residues is not required for chemotactic activity. Given this chemotactic capability, it is possible that BGP acts in bone remodelling by attracting osteogenic cells to the sites of bone resorption where BGP may be liberated or exposed.     

Bone-derived macrophage chemotactic factors: Methods of extraction and further characterization

Summary Mononuclear phagocytes have been implicated as important cellular elements in the process of bone resorption. We have postulated that the recruitment and migration of mononuclear phagocytes to bone occurs via a mechanism(s) in which bone-derived chemotactic factors (BDCF) are released from foci undergoing resorption. In the experiments presented here we have used newborn mouse calvaria and examined a variety of extraction protocols, both dissociative and nondissociative, as means of obtaining stable and reproducible chemotactic activity for mouse peritoneal macrophages. Chemotaxis and chemokinesis were assessed using a multiwell chamber modification of the Boyden transfilter method. Further, we have attempted to purify the BDCF by both molecular sieve and anion exchange chromatography. Our results indicated that nondissociative extraction with 0.5 M EDTA in the presence of 1% DMSO yielded the most potent and reproducible chemotactic activity. The results of molecular sieve and anion exchange chromatography suggested that there were several BDCF activities in these preparations and that their molecular weights were probably in the range of from 14,000–67,000 daltons. Anion exchange chromatography also demonstrated the presence of a fraction, eluted with 2 M NaCl, with high chemotactic activity and minimal protein concentration. These observations confirmed the suggestion that there are several macrophage chemotactic factors in bone which have as yet to be identified, and suggest methods for pursuing their isolation.     

Cigarette smoke extract inhibits chemotaxis and collagen gel contraction mediated by human bone marrow osteoprogenitor cells and osteoblast-like cells

Cell migration and matrix remodeling are key events in tissue repair and restructuring. Osteoblasts are responsible for the production of new bone matrix during bone remodeling. The activity of these cells can be modulated by a number of factors. The current study evaluated the hypothesis that cigarette smoke extract can alter repair and remodeling responses of human osteoprogenitor cells and osteoblast-like cells and, therefore, could explain one mechanism by which cigarette smoking leads to osteoporosis. Human osteoprogenitor cells were isolated from normal human bone marrow and maintained in culture under either control conditions or conditions that induced differentiation into osteoblast-like cells. Both cell types migrated toward fibronectin and PDGF-BB as chemoattractants. Neither responded to TGF-beta1. The osteoprogenitor cells were more active in their chemotactic response. The chemotactic response of both cell types was inhibited by cigarette smoke extract in a concentration-dependent manner. Both cell types, when cultured in three-dimensional native collagen gels maintained in floating culture, induced contraction of their surrounding matrices. Contraction was augmented by serum, PDGF-BB, and TGF-beta1. Osteoprogenitor cells were less active in inducing contraction than were osteoblast-like cells. Contraction of both cell types was inhibited by cigarette smoke extract. Cigarette smoke extract also inhibited the production of fibronectin by both cell types maintained in three-dimensional culture. Addition of exogenous fibronectin partially restored the ability of the cells to contract three-dimensional collagen gels. The current study demonstrates that cigarette smoke can interfere with the ability of bone cells to participate in repair and remodeling events. Such an effect may be one mechanism leading to the development of osteoporosis.     

Localization of bone morphogenetic protein 13 in human intervertebral disc and its molecular and functional effects in vitro in 3D culture

Our laboratory has demonstrated that bone morphogenetic protein 13 prevented the effects of annular injury in an ovine model, maintaining intervertebral disc height, cell numbers and increasing extracellular matrix production compared to degenerated controls. The present study sought to examine the molecular effects of bone morphogenetic protein 13 on human degenerated disc cells and localize its expression in both human degenerate and scoliotic disc tissue. Effect of bone morphogenetic protein 13 on human derived nucleus pulposus, annulus fibrosus and endplate cells cultured in alginate beads was evaluated by changes in proteoglycan and collagen content. Migratory potential of disc cells towards bone morphogenetic protein 13 was also examined. Bone morphogenetic protein 13 induced significant proteoglycan accumulation in nucleus (18%), annulus (21%) and endplate (23%) cells cultured in alginate beads (p < 0.05) compared to controls. Further bone morphogenetic protein 13 increased collagen I and II protein expression in nucleus and endplate cells. Nucleus cells displayed a significant chemotactic response towards bone morphogenetic protein 13. The endogenous expression of bone morphogenetic protein 13 in degenerate disc tissue was not different to scoliotic disc. Bone morphogenetic protein 13 has the potential to enhance extracellular matrix accumulation and induce cell migration in certain disc cells. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1769–1775, 2015.     

Effects of the proteinase inhibitors leupeptin and E-64 on osteoclastic bone resorption

Summary To determine the possible involvement of cysteine-proteinases in bone matrix degradation by osteoclasts, the effects of the proteinase inhibitors leupeptin and E-64 were studied in anin vitro system using mouse bone explants. It was observed that in explants treated with the drugs, the amount of demineralized matrix opposing the ruffled border of the osteoclasts increased about 20-fold within 6 hours. This suggests that demineralization had proceeded whereas matrix degradation had been retarded. It was further noticed that in 12 of 287 osteoclasts, cytoplasmic vacuoles were present containing collagen fibrils that could not be distinguished from those in cartilage or bone. Their intracellular localization was proved by the study of serial sections. Finally, a significant reduction was shown as to the relative surface density of electrontranslucent vacuoles; this would seem to suggest reduced endocytic activity of the cells. Our observations support the view that cysteine-proteinases play an important role in osteoclastic bone resorption. It was further noticed that thein vitro effects of leupeptin and E-64 in certain respects resemble ultrastructural features of pycnodysostosis, an osteopetrosislike bone disorder. The data are in line with the hypothesis that this disease is caused by insufficient activity of osteoclastic cysteine-proteinases.     

Cytochemical studies of the cellular events sequence in bone remodeling: Cytological evidence for a coupling mechanism

In order to elucidate the sequence of the cellular events in the bone remodeling process which may contribute to the coupling mechanism between bone resorption and formation, this study was attempted to observe the cytochemical localization of acid phosphatase (ACPase) and alkaline phosphatase (ALPase) activities in the remodeling bone cells. The ACPase activity in osteoclasts could be demonstrated in the vacuoles, extracellular channels of the ruffled border and spaces between cells and bone. In addition, the reaction product was observed at the resorbed bone surface, which was continued to the cement line resulting in new bone formation. Attached to the resorbed bone surface and newly formed cement line, ALPase-positive osteoblastic cells appeared and formed new bone matrix. These suggest that osteoclasts may move as resorbing bone and form a new cement line, which may be responsible for coupling of bone resorption and formation. Moreover, the ACPase-positive mononuclear cells could be observed on the resorbed bone surface adjacent to the resorbing osteoclast. They were characterized by numerous cytoplasmic extensions, sparse r-ER and mitochondria, and many lysosomes. However, the osteocytes being excavated by osteoclasts also showed the similar appearance. In the cytochemical study, these osteocytes showed ACPase activity in lysosomes in the same way as the mononuclear cells on the resorbed bone surface. Therefore, it is suggested that the ACPase-positive mononuclear cells principally originate from the osteocytes which are excavated by osteoclasts from bone matrix, and these cells are identified with the mononuclear phagocytes in reversal phase which have been postulated.     

Infrared spectroscopy indicates altered bone turnover and remodeling activity in renal osteodystrophy

Renal osteodystrophy alters metabolic activity and remodeling rate of bone and also may lead to different bone composition. The objective of this study was to characterize the composition of bone in high‐turnover renal osteodystrophy patients by means of Fourier transform infrared spectroscopic imaging (FTIRI). Iliac crest biopsies from healthy bone (n = 11) and patients with renal osteodystrophy (ROD, n = 11) were used in this study. The ROD samples were from patients with hyperparathyroid disease. By using FTIRI, phosphate‐to‐amide I ratio (mineral‐to‐matrix ratio), carbonate‐to‐phosphate ratio, and carbonate‐to‐amide I ratio (turnover rate/remodeling activity), as well as the collagen cross‐link ratio (collagen maturity), were quantified. Histomorphometric analyses were conducted for comparison. The ROD samples showed significantly lower carbonate‐to‐phosphate (p < .01) and carbonate‐to‐amide I (p < .001) ratios. The spatial variation across the trabeculae highlighted a significantly lower degree of mineralization (p < .05) at the edges of the trabeculae in the ROD samples than in normal bone. Statistically significant linear correlations were found between histomorphometric parameters related to bone‐remodeling activity and number of bone cells and FTIRI‐calculated parameters based on carbonate‐to‐phosphate and carbonate‐to‐amide I ratios. Hence the results suggested that FTIRI parameters related to carbonate may be indicative of turnover and remodeling rate of bone. © 2010 American Society for Bone and Mineral Research     

Calvarial Osteoclasts Express a Higher Level of Tartrate-Resistant Acid Phosphatase than Long Bone Osteoclasts and Activation Does not Depend on Cathepsin K or L Activity

Bone resorption by osteoclasts depends on the activity of various proteolytic enzymes, in particular those belonging to the group of cysteine proteinases. Next to these enzymes, tartrate-resistant acid phosphatase (TRAP) is considered to participate in this process. TRAP is synthesized as an inactive proenzyme, and in vitro studies have shown its activation by cysteine proteinases. In the present study, the possible involvement of the latter enzyme class in the in vivo modulation of TRAP was investigated using mice deficient for cathepsin K and/or L and in bones that express a high (long bone) or low (calvaria) level of cysteine proteinase activity. The results demonstrated, in mice lacking cathepsin K but not in those deficient for cathepsin L, significantly higher levels of TRAP activity in long bone. This higher activity was due to a higher number of osteoclasts. Next, we found considerable differences in TRAP activity between calvarial and long bones. Calvarial bones contained a 25-fold higher level of activity than long bones. This difference was seen in all mice, irrespective of genotype. Osteoclasts isolated from the two types of bone revealed that calvarial osteoclasts expressed higher enzyme activity as well as a higher level of mRNA for the enzyme. Analysis of TRAP-deficient mice revealed higher levels of nondigested bone matrix components in and around calvarial osteoclasts than in long bone osteoclasts. Finally, inhibition of cysteine proteinase activity by specific inhibitors resulted in increased TRAP activity. Our data suggest that neither cathepsin K nor L is essential in activating TRAP. The findings also point to functional differences between osteoclasts from different bone sites in terms of participation of TRAP in degradation of bone matrix. We propose that the higher level of TRAP activity in calvarial osteoclasts compared to that in long bone cells may partially compensate for the lower cysteine proteinase activity found in calvarial osteoclasts and TRAP may contribute to the degradation of noncollagenous proteins during the digestion of this type of bone. An erratum to this article is available at .     

Induction of human osteoprogenitor chemotaxis, proliferation, differentiation, and bone formation by osteoblast stimulating factor-1/pleiotrophin: osteoconductive biomimetic scaffolds for tissue engineering.

The process of bone growth, regeneration, and remodeling is mediated, in part, by the immediate cell-matrix environment. Osteoblast stimulating factor-1 (OSF-1), more commonly known as pleiotrophin (PTN), is an extracellular matrix-associated protein, present in matrices, which act as targets for the deposition of new bone. However, the actions of PTN on human bone progenitor cells remain unknown. We examined the effects of PTN on primary human bone marrow stromal cells chemotaxis, differentiation, and colony formation (colony forming unit-fibroblastic) in vitro, and in particular, growth and differentiation on three-dimensional biodegradable porous scaffolds adsorbed with PTN in vivo. Primary human bone marrow cells were cultured on tissue culture plastic or poly(DL-lactic acid-co-glycolic acid) (PLGA; 75:25) porous scaffolds with or without addition of recombinant human PTN (1 pg-50 ng/ml) in basal and osteogenic conditions. Negligible cellular growth was observed on PLGA scaffold alone, generated using a super-critical fluid mixing method. PTN (50 microg/ml) was chemotactic to human osteoprogenitors and stimulated total colony formation, alkaline phosphatase-positive colony formation, and alkaline phosphatase-specific activity at concentrations as low as 10 pg/ml compared with control cultures. The effects were time-dependent. On three-dimensional scaffolds adsorbed with PTN, alkaline phosphatase activity, type I collagen formation, and synthesis of cbfa-1, osteocalcin, and PTN were observed by immunocytochemistry and PTN expression by in situ hybridization. PTN-adsorbed constructs showed morphologic evidence of new bone matrix and cartilage formation after subcutaneous implantation as well as within diffusion chambers implanted into athymic mice. In summary, PTN has the ability to promote adhesion, migration, expansion, and differentiation of human osteoprogenitor cells, and these results indicate the potential to develop protocols for de novo bone formation for skeletal repair that exploit cell-matrix interactions.     

Matrix Concentration of Insulin-like Growth Factor I (IGF-I) is Negatively Associated with Biomechanical Properties of Human Tibial Cancellous Bone Within Individual Subjects

Insulin-like growth factor-I (IGF-I), abundant in bone matrix, is believed to play an important role during bone development and remodeling. To our knowledge, however, few studies have addressed the relationship between the concentration of IGF-I in bone matrix and the biomechanical properties of bone tissue. In this study, forty-five cylindrical specimens of cancellous bone were harvested from six human tibiae and scanned using microcomputed tomography (μCT). The bone volume fraction (BV/TV) was calculated from three-dimensional (3D) μCT images. Mechanical tests were then performed on a servohydraulic testing system to determine the strength and stiffness of cancellous bone. Following mechanical testing, the concentration of IGF-I in bone matrix was measured by using an enzyme-linked immunoabsorbent assay (ELISA). Within each subject, the concentration of IGF-I in bone matrix had significant (P < 0.01) negative correlations with the bone volume fraction, strength, and stiffness of cancellous bone. In particular, the anterior quadrant of the proximal tibia was significantly (P < 0.02) greater in IGF-I matrix concentration and marginally significantly lower in strength (P = 0.053) and stiffness (P = 0.059) than the posterior quadrant. The negative correlations between the cancellous bone matrix concentration of IGF-I and cancellous bone biomechanical properties within subjects found in this study may help us understand the variation of the biomechanical properties of cancellous bone in proximal human tibiae.     

Osteogenesis by human osteoblastic cells in diffusion chamber In vivo

Osteogenic potential of osteoblastic cells isolated from human bone was evaluated by a diffusion chamber method. Cells placed in diffusion chambers were implanted intraperitcneally into the athymic mice. The diffusion chambers cultured in vivo were harvested and examined after implantation for 6–8 weeks. The content of the chamber was proved by a soft roentogenogram to contain the radioopaque area. Light microscopic study revealed that this area was made up of bone-like nodule. Within the diffusion chambers, the cell layers were observed alongside the interior surface of the membrane filters, and mineralized nodules were formed among the cell layers. The mineralized nodules were confirmed by staining with the von Kossa technique for calcium mineral deposits. In electron microscopic study, the osteoblastic cells had a relatively large nuclei, and an abundant rough endoplasmic reticulum produced numerous extracellular matrix. In the bone-like nodules, the osteoblastic cells were surrounded by a heavily mineralized matrix with nonmineralized matrix separating the osteoblastic cells from the mineralized matrix. The mineralized matrix contained well-banded collagen fibrils. Matrix vesicles and collagen fibrils which were closely associated with mineral deposition were observed at the mineralizing front. These results together with our previous report [13] indicate that isolated human osteoblastic cells possessed osteogenic potential in vivo as well as mineralization activity in vitro.     

The distribution of trace metal ions in bone and tendon

The natural trace metal compositions of human bone mineral and demineralized bone were measured by emission spectroscopy and compared with those of the original whole bone and human tendon. Cu, Fe and Zn remained in the collagenous matrix of bone and were found in similar quantities in tendon. It is suggested, therefore, that these ions are chemically bound to the collagen matrix in these tissuesin vivo, and that the binding has a part in their activity. Most of the Pb, Si, Sr, and V in bone remained with the mineral portion, probably as substituted or interstitial ions. Zn is divided between the organic and mineral phases.This work was supported in part by Grant No. AMO 7626, National Institutes of Health, United States Public Health Service and by the Veterans Administration Research Service.     

Osteoid induction

Osteoinduction is a biological principle. The implantation of tissue with inductive properties results in the proliferation and differentiation of undifferentiated cells to cartilage and bone. This process, which is similar to a cascade-type mechanism, is controlled by a series of humoral and local growth factors. It was possible to isolate a number of macromolecular substances with osteoinductive, mitogenic, or chemotactic properties specifically from the extracellular bone matrix. A deeper understanding of the regulative mechanisms as well as the greater availability of growth factors may lead to new therapeutic approaches in bone surgery.