Human Bone Explants Research Articles

Pharmacological elevation of sphingosine-1-phosphate by S1P lyase inhibition accelerates bone regeneration after post-traumatic osteomyelitis.

Posttraumatic osteomyelitis and the ensuing bone defects are a debilitating complication after open fractures with little therapeutic options. We have recently identified potent osteoanabolic effects of sphingosine-1-phosphate (S1P) signalling and have now tested whether it may beneficially affect bone regeneration after infection. We employed pharmacological S1P lyase inhibition by 4-deoxypyrodoxin (DOP) to raise S1P levels in vivo in an unicortical long bone defect model of posttraumatic osteomyelitis in mice. In a translational approach, human bone specimens of clinical osteomyelitis patients were treated in organ culture in vitro with DOP. Bone regeneration was assessed by μCT, histomorphometry, immunohistology and gene expression analysis. The role of S1P receptors was addressed using S1PR3 deficient mice. Here, we present data that DOP treatment markedly enhanced osteogenesis in posttraumatic osteomyelitis. This was accompanied by greatly improved osteoblastogenesis and enhanced angiogenesis in the callus accompanied by osteoclast-mediated bone remodelling. We also identified the target of increased S1P to be the S1PR3 as S1PR3-/- mice showed no improvement of bone regeneration by DOP. In the human bone explants, bone mass significantly increased along with enhanced osteoblastogenesis and angiogenesis. Our data suggest that enhancement of S1P/S1PR3 signalling may be a promising therapeutic target for bone regeneration in posttraumatic osteomyelitis.

Ex vivo determination of chitosan/curdlan/hydroxyapatite biomaterial osseointegration with the use of human trabecular bone explant: New method for biocompatibility testing of bone implants reducing animal tests

Permanent orthopedic/dental implants should reveal good osseointegration, which is defined as an ability of the biomaterial to form a direct connection with the surrounding host bone tissue after its implantation into the living organism. Currently, biomaterial osseointegration is confirmed exclusively with the use of in vivo animal tests. This study presents for the first time ex vivo determination of osseointegration process using human trabecular bone explant that was drilled and filled with the chitosan/curdlan/hydroxyapatite biomaterial, followed by its long-term culture under in vitro conditions. Within this study, it was clearly proved that tested biomaterial allows for the formation of the connection with bone explant since osteoblasts, having ability to produce bone extracellular matrix (type I collagen, fibronectin), were detected at a bone-implant interface by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Importantly, in this research it was demonstrated by Live/Dead staining and CLSM imaging that human bone explants may stay alive for a long period of time (at least approx. 50 days) during their culture under in vitro conditions. Therefore, ex vivo bone explant, which is a heterogeneous tissue containing many different cell types, may serve as an excellent model to test biomaterial osseointegration during comparative and preliminary studies, reducing animal tests which is compatible with the principles of ‘3Rs’, aiming to Replace, Reduce and Refine the use of animals wherever possible.

Human somatic cell mutagenesis creates genetically tractable sarcomas.

Creating spontaneous yet genetically tractable human tumors from normal cells presents a fundamental challenge. Here we combined retroviral and transposon insertional mutagenesis to enable cancer gene discovery starting with human primary cells. We used lentiviruses to seed gain- and loss-of-function gene disruption elements, which were further deployed by Sleeping Beauty transposons throughout the genome of human bone explant mesenchymal cells. De novo tumors generated rapidly in this context were high-grade myxofibrosarcomas. Tumor insertion sites were enriched in recurrent somatic copy-number aberration regions from multiple cancer types and could be used to pinpoint new driver genes that sustain somatic alterations in patients. We identified HDLBP, which encodes the RNA-binding protein vigilin, as a candidate tumor suppressor deleted at 2q37.3 in greater than one out of ten tumors across multiple tissues of origin. Hybrid viral-transposon systems may accelerate the functional annotation of cancer genomes by enabling insertional mutagenesis screens in higher eukaryotes that are not amenable to germline transgenesis.

Targeting bone alleviates osteoarthritis in osteopenic mice and modulates cartilage catabolism.

ObjectiveSubchondral bone modifications occur early in the development of osteoarthritis (OA). The level of bone resorption might impact cartilage remodeling. We therefore assessed the in vivo and in vitro effects of targeting bone resorption in OA and cartilage metabolism.MethodsOA was induced by meniscectomy (MNX) in ovariectomized osteopenic mice (OP) treated with estradiol (E2), pamidronate (PAM), or phosphate buffered saline (PBS) for 6 weeks. We assessed the subchondral bone and cartilage structure and the expression of cartilage matrix proteases. To assess the involvement of bone soluble factors in cartilage metabolism, supernatant of human bone explants pre-treated with E2 or PAM were transferred to cartilage explants to assess proteoglycan release and aggrecan cleavage. OPG/RANKL mRNA expression was assessed in bone explants by real-time quantitative PCR. The role of osteoprotegerin (OPG) in the bone-cartilage crosstalk was tested using an OPG neutralizing antibody.ResultsBone mineral density of OP mice and osteoclast number were restored by E2 and PAM (p<0.05). In OP mice, E2 and PAM decreased ADAMTS-4 and -5 expression, while only PAM markedly reduced OA compared to PBS (2.0±0.63 vs 5.2±0.95; p<0.05). OPG/RANKL mRNA was increased in human bone explants treated with both drugs (2.2–3.7-fold). Moreover, supernatants from bone explants cultured with E2 or PAM reduced aggrecan cleavage and cartilage proteoglycan release (73±8.0% and 80±22% of control, respectively, p<0.05). This effect was reversed with osteoprotegerin blockade.ConclusionThe inhibition of bone resorption by pamidronate in osteopenic mice alleviates the histological OA score with a reduction in the expression of aggrecanases. Bone soluble factors, such as osteoprotegerin, impact the cartilage response to catabolic factors. This study further highlights the importance of subchondral bone in the regulation of joint cartilage damage in OA.

Bone Microbial Decontamination Agents in Osseous Grafting: An In Vitro Study With Fresh Human Explants

Establishing a safe prophylactic antimicrobial protocol in bone grafting may enhance osseous volume outcomes. The purpose of this in vitro study is to assess human osteoblast response and safety after explant antimicrobial exposure. Fresh human bone explants were exposed to three antimicrobials: povidone-iodine (PovI; 0.05%, 1%, and 5%), chlorhexidine (CHX; 0.2% and 1%), and sodium hypochlorite (NaOCl; 2.5%, 4.5%, and 5.25%) at different times (15, 30, 45, and 60 seconds) and concentrations to assess cellular toxicity. Explants were washed three times with saline after exposure. Controls, explants cultured in the absence of antimicrobials, were performed for all experimental situations tested. Trials were conducted in triplicate. Particle size influence on osteoblast growth was determined between bone fragments with a diameter <2 and ≥2 to 5 mm. Test and control groups were monitored by light microscopy to evaluate cellular growth. Osteoblast differentiation and morphology was assessed by alkaline phosphatase activity and scanning electron microscopy (SEM). Osteoblast growth was similar for particles <2 and ≥2 to 5 mm. Alkaline phosphatase control reference values were not significantly different from test groups (0.35 mU/mL ± 0.004 versus 0.34 mU/mL ± 0.009; P >0.05). Light microscopy showed on average 97% osteoblastic growth for bone particles exposed to PovI 5% and CHX 0.2% for all times and CHX 1% up to 30 seconds. The odds ratio of positive osteoblastic growth after a 30-second 2.5% NaOCl exposure was 2.4 times higher than after 5.25%. On average, one of two replicas yielded positive growth with 2.5% NaOCl and one of three with 5.25%. After 60-second explant exposure, positive osteoblastic growth was 7.7 times more likely to occur with 5% PovI or 0.2% CHX than with 5.25% NaOCl (P <0.05). SEM analysis confirmed light microscopy similar cellular adhesion and osteoblast phenotypic features between test and control groups. Best osteoblastic growth occurred after bone PovI exposure and CHX 0.2%. Cellular toxicity seems to be influenced by the type of antimicrobial, concentration, and exposure time. SEM analysis confirmed absence of osteoblast phenotypic alterations after exposure. Decontamination agents can safely be used in bone transplantation using up to 5% PovI and 0.2% CHX for 1 minute and CHX 1% for 30 seconds.

Expression of smooth muscle actin in osteoblasts in human bone

It is well known that certain connective tissue cells (viz., dermal fibroblasts) can express the gene for a muscle actin – α-smooth muscle actin – and can contract. This process contributes to skin wound closure and is responsible for Dupuytren's contracture. The objective of this study was to determine if human osteoblasts can also express the gene for α-smooth muscle actin. Immunohistochemistry using a monoclonal antibody for α-smooth muscle actin was performed on human cancellous bone samples obtained from 20 individuals at the time of total joint arthroplasty. The percentages of resting and active osteoblasts on the bone surfaces containing this muscle actin isoform were evaluated. Explants of human bone were also studied for the expression of α-smooth muscle actin in the tissue and in the outgrowing cells with time in culture. Western blot analysis was performed to quantify the α-smooth muscle actin content of the outgrowing cells relative to smooth muscle cell controls. Nine ± 2% (mean ± SEM; n=20) of the cells classified as inactive osteoblasts and 69±3% ( n=19) of the cells identified as active osteoblasts on the bone surface contained α-smooth muscle actin. This difference was highly statistically significant (Student's t test, p<0.0001). Similar profiles of α-smooth muscle actin-expressing cells were found in explants cultured for up to 12 weeks. Cells forming a layer on the surface of the explants and growing out from them in monolayer also contained α-smooth muscle actin by immunohistochemistry and Western blot analysis. Human osteoblasts can express the gene for α-smooth muscle actin. This expression should be considered a phenotypic characteristic of this cell type, conferred by its progenitor cells: bone marrow stromal-derived stem cells, and perhaps pericytes and smooth muscle cells.

Inorganic phosphate induces apoptosis of osteoblast-like cells in culture

The major goal of this investigation was to test the hypothesis that one of the major products of bone resorption, inorganic phosphate (Pi), activates osteoblast apoptosis. Osteoblast-like cells were isolated from explants of human bone. In monolayer culture, these cells showed an osteogenic phenotype. Thus, the cells exhibited raised alkaline phosphatase activity, expressed osteogenic messenger RNA transcripts, and formed biological mineral. When these cells were treated with 1–7 mmol/L Pi there was a dose- and time-dependent decrease in cell viability. Accordingly, after 48 h, 5 mmol/L Pi reduced the number of viable osteoblast-like cells by 25%; 7 mmol/L Pi reduced the number of cells by 60%. By 96 h, following treatment with 5 mmol/L Pi, the percentage of viable cells was 30%, whereas 7 mmol/L Pi caused an almost complete loss of osteoblast viability. Osteoblast death was blocked by treating the cells with phosphonoformic acid, an inhibitor of the plasma-membrane Na-Pi transporter. Using morphological and end-labeling procedures, we confirmed that cell death was through apoptosis. To probe the mechanism of cell death, osteoblast-like cells were probed with rhodamine 123, a dye that is responsive to the membrane potential. We noted that Pi-treated cells displayed a profound loss of mitochondrial membrane potential, suggesting that the anion activated the death program through the induction of a mitochondrial membrane permeability transition. We conclude that high levels of osteoblast apoptosis observed at sites of bone resorption may be linked to release of Pi from bone mineral.

CKbeta-8 [CCL23], a novel CC chemokine, is chemotactic for human osteoclast precursors and is expressed in bone tissues.

We have previously demonstrated that a tartrate-resistant acid phosphatase (TRAP)-positive subpopulation of mononuclear cells isolated from collagenase digests of human osteoclastoma tissue exhibits an osteoclast phenotype and can be induced to resorb bone. Using these osteoclast precursors as a model system, we have assessed the chemotactic potential of 16 chemokines. Three CC chemokines, the recently described CKbeta-8, RANTES, and MIP-1alpha elicited significant chemotactic responses. In contrast, 10 other CC chemokines (MIP-1beta, MCP-1, MCP-2, MCP-3, MCP-4, HCC-1, eotaxin-2, PARC, SLC, ELC) and 3 CXC chemokines (IL-8, GROalpha, SDF-1) were inactive. None of these chemokines showed any chemotactic activity for either primary osteoblasts derived from human bone explants or the osteoblastic MG-63 cell line. The identity of the osteoclast receptor that mediates the chemotactic response remains to be established. However, all three active chemokines have been reported to bind to CCR1 and cross-desensitization studies demonstrate that RANTES and MIP-1alpha can partially inhibit the chemotactic response elicited by CKbeta-8. CKbeta-8, the most potent of the active CC chemokines (EC(max) 0.1-0.3 nM), was further characterized with regard to expression in human bone and cartilage. Although expression is not restricted to these tissues, CKbeta-8 mRNA was shown to be highly expressed in osteoblasts and chondrocytes in human fetal bone by in situ hybridization. In addition, CKbeta-8 protein was shown to be present in human osteophytic tissue by immunolocalization. These observations suggest that CKbeta-8, and perhaps other chemokines, may play a role in the recruitment of osteoclast precursors to sites of bone resorption.

Advanced glycation endproducts stimulate interleukin-6 production by human bone-derived cells.

Advanced glycation endproducts (AGEs), which result from nonenzymatic reactions of glucose with tissue proteins, have been shown to accumulate on long-lived proteins in advanced aging and diabetes mellitus. Thus, AGEs have been implicated in some of the chronic complications associated with these disorders. In this study, we investigated the effects of the glucose-modified protein on the production of the potent bone resorption factors by cells derived from explants of human bone. AGEs stimulated the release of interleukin-6 (IL-6) in the culture supernatants from the bone-derived cells and increased the levels of IL-6 mRNA in the cells. By contrast, the levels of IL-11 in the culture supernatants were not altered by AGEs, and the other bone resorption factors IL-1 alpha and IL-1 beta were undetectable (< 1.0 pg/ml) either without or with the treatment of AGEs. Electrophoretic mobility-shift assays revealed that the transcription nuclear factor-kappa B, which is critical for the inducible expression of IL-6, was activated in the nuclear extracts from mouse osteoblastic MC3T3-E1 cells treated with AGEs. These results suggest that AGEs are involved in bone remodeling modulation by stimulating IL-6 production in human bone-derived cells.

Role of protein kinase C alpha, Arf, and cytoplasmic calcium transients in phospholipase D activation by sodium fluoride in osteoblast-like cells.

The effect of fluoride on phospholipase D (PLD) activation was studied using in vitro culture of Saos-2, MG-63 osteosarcoma cells, and normal osteoblast-like cells derived from human bone explants. Millimolar concentrations of NaF induced a significant accumulation of phosphatidylethanol (PEt) in Saos-2 cells but not in MG-63 and normal osteoblast-like cells. PLD activation was evident at 15 mM and concentration-dependent up to 50 mM. This stimulation was inhibited by deferoxamine, a chelator of Al3+, suggesting that PLD activation involves fluoride-sensitive G proteins. A good correlation was found between the levels of intracellular free Ca2+ and the activation of PLD. The time courses of the two responses were nearly identical. The ability of NaF to induce both responses was largely dependent on the presence of extracellular calcium. The calcium ionophore A23187 reproduced the effect of NaF, and this effect was antagonized by EGTA, suggesting that PLD activation was, at least in part, a calcium-regulated event. Phorbol 12-myristate 13-acetate (PMA) also stimulated PLD activity in human bone cells. Protein kinase C alpha (PKC alpha) and epsilon were expressed in Saos-2 cells. Acute pretreatment of cells with PMA reduced concomitantly the amounts of PKC alpha, but not of PKC epsilon, and the subsequent activation of PLD elicited by PKC activators. The PLD response to NaF was not attenuated but rather enhanced by down-regulation of PKC alpha. Therefore, PKC-alpha-induced PLD activation is unlikely to mediate the effect of NaF. Moreover, PMA and NaF showed a supraadditive effect on PLD activation in Saos-2 cells. This stimulation, in contrast to NaF alone, was not reduced by EGTA. Hence, mobilization of calcium by NaF cannot account for the enhanced PLD activation in response to PMA stimulation. Membrane Arf and RhoA contents were assessed by Western immunoblot analyses. Membranes derived from NaF-stimulated Saos-2 cells contained more Arf and RhoA when compared with membranes derived from control or PMA-stimulated cells. Translocation of the small GTPases was calcium-independent. We conclude that PLD activation by NaF in Saos-2 cells includes a fluoride-sensitive G protein, increases in the levels of intracellular calcium, and Arf/RhoA redistribution to membranes. The results also indicate that the NaF-induced Arf/RhoA translocation exerts in concert with PMA-activated PKC alpha a synergistic effect on the activation of PLD in Saos-2 cells.

Expression and secretion of parathyroid hormone-related protein by human bone-derived cells in vitro: Effects of glucocorticoids

We investigated the production of parathyroid hormone-related protein (PTHrP) by cells derived from explants of human bone. Using an immunoradiometric assay (IRMA), PTHrP was detected in conditioned medium from cultures of bone-derived cells from 6 of 7 patients investigated in this study. PTHrP mRNA was identified in human bone cells using reverse transcriptase-linked polymerase chain reaction (RT-PCR) and by Northern analysis. Transcripts for PTHrP were detected in a purified population of alkaline phosphatase positive cells isolated from human bone marrow cultures by flow cytometry, confirming the expression of PTHrP mRNA by cells of the osteoblastic lineage. Production of PTHrP was inhibited by 10(-6) M of the glucocorticoids, prednisolone and desacetylated deflazacort, in a dose-dependent manner. In addition, RT-PCR followed by Southern blot analysis detected a decrease in steady-state PTHrP mRNA in cultures of human bone-derived cells treated with 10(-6) M prednisolone.

Ultrastructure of human osteoblasts and associated matrix in culture.

The ultrastructure, as visualized by transmission electron microscopy, of cells obtained from human bone explants and subsequently cultured is described along with the electron microscopic appearance of the associated intercellular matrix. The cells were characterized as osteoblasts on the basis of immunohistochemical, enzymatic, and functional criteria. Although the osteoblasts could be cultured in standard culture media and always appeared singly, not forming syncytia, the cultures were eventually confluent and formed multilayers. The cells were fusiform or cuboidal with diameters ranging between 10-15 microns. The cytoplasm was characterized by numerous large mitochondria, and especially by a very prominent RER. The intercellular matrix was woven with collagen fibres surrounding large numbers of matrix vesicles. In areas with matrix vesicles, evidence for osteoblast activity, i.e. mineralization related to matrix vesicles, could be observed after incubation with beta-glycerophosphate. In conclusion, we provide evidence that human osteoblasts cultured in vitro synthesize collagen and produce a matrix with vesicles capable of initiating mineralization processes.

Human osteoblasts synthesize and respond to platelet-derived growth factor

Platelet-derived growth factor (PDGF) is a potent mitogen for cells of mesenchymal origin. We previously demonstrated that PDGF is produced by osteogenic sarcoma cells. We report here that normal human bone-derived cells produce PDGF and that these cells have an osteoblastic phenotype. This was demonstrated by use of a double immunofluorescent technique and by examining cloned human adult osteoblasts. Northern blot analysis indicates that PDGF production is accounted for by expression of the PDGF-A gene. PDGF-AA and PDGF-BB generally stimulated thymidine incorporation in normal human bone explants to a similar extent. All of the cloned human osteoblasts responded to PDGF-BB while the response to PDGF-AA varied. Similarly, five cloned osteoblastic cell populations were shown to produce PDGF while one did not. This result supports the hypothesis that there are different osteoblastic cell populations that differ in their growth factor responses or in the production of growth factors. Our results suggest that PDGF-BB has the potential to act as a paracrine factor for normal human osteoblasts because all of the osteoblastic cell populations responded to PDGF-BB. None of the osteoblastic cell populations expressed the PDGF-B gene, indicating that it would not act as an autocrine factor. Although not definitive, our results suggest that PDGF-AA has the potential to act as an autocrine factor because osteoblastic bone cell populations were shown to express the PDGF-A gene and respond to PDGF-AA.

Influence of experimental conditions on osteoblast activity in human primary bone cell cultures

Primary bone cell cultures were derived from human bone explants. Cellular activity was characterized by the alkaline phosphatase (AP) activity, osteocalcin, and type I and III collagen secretions in the supernatant. The determination of bone cell activity was performed in three different wells. No significant difference was noted between wells: the coefficient of variation was 8.0 +/- 2.9% for AP activity, 18.3 +/- 1.9% for osteocalcin secretion, and 22.5 +/- 14.3% for collagen. The AP activity and osteocalcin secretion significantly decreased with the number of passages: they were the highest after the first passage. Between each subject, the coefficient of variation was 85% for AP activity and osteocalcin secretion and 63 and 57% for type I and III collagen secretion, respectively. The AP activity did not differ with the age or sex of the donor. In contrast, osteocalcin secretion was significantly lower in females than in males. In males, osteocalcin significantly decreased with the age of the donor (r = -0.61; p less than 0.05). Cellular activity did not depend on the site of the biopsy. When bone explants from one donor were cultured in two different petri dishes, the activity of cells was similar in both dishes, except in one case. Primary cell cultures derived from human bone explants are the only model providing untransformed osteoblastlike cells of human origin. Because of the experimental conditions, some factors may have influenced the cellular activity and they must be taken into account to validate further in vitro studies.

Hereditary resistance to 1,25-dihydroxyvitamin D: defective function of receptors for 1,25-dihydroxyvitamin D in cells cultured from bone.

The syndrome of rickets, alopecia, hypocalcemia, and high circulating levels of 1,25-dihydroxyvitamin D (1,25-(OH)2D) apparently is caused by resistance of target tissues to 1,25-(OH)2D. To evaluate this, we cultured cells from explants of long bone of one patient with this syndrome and from a control without any preexisting disorder of mineral metabolism. The cultured cells showed morphological features of fibroblasts but contained alkaline phosphatase activity without detectable acid phosphatase activity, indicating an osteoblastic origin for some or all of the cultured cells. Receptors for 1,25-(OH)2D were assessed by three methods: high affinity uptake of hormone in nuclei of dispersed cells, high affinity binding in hypertonic extracts (herein termed cytosol) from cells, and sedimentation velocity of bound [3H]1,25-(OH)2D3 in extracts of cell nuclei. With cells cultured from bone of the normal control, receptors for 1,25-(OH)2D exhibited properties indistinguishable from those found with cultured skin fibroblasts. With cells cultured from bone of the patient with resistance to 1,25-(OH)2D, high affinity uptake of 1,25-(OH)2D into nuclei was unmeasurable, but high affinity binding of hormone with cytosol was normal; these abnormal findings also were indistinguishable from abnormal findings obtained with fibroblasts cultured from skin of that patient. 1) Cells cultured from explants of human bone showed morphological features of fibroblasts but retained a marker enzyme characteristic of osteoblasts. Significant admixture of osteoblast-like cells with fibroblasts was possible. 2) Cells cultured from bone of a patient with familial resistance to 1,25-(OH)2D exhibit a defect in vitamin D metabolism, indistinguishable from the defect observed with cells cultured from skin of the same patient.