In vitro testing of Advanced JAX Bone Void Filler System: species differences in the response of bone marrow stromal cells to beta tri-calcium phosphate and carboxymethylcellulose gel.

The Advanced JAX Bone Void Filler System (AJBVFS) is a novel bone graft material manufactured by Smith and Nephew Orthopaedics Ltd. and comprises beta tri-calcium phosphate granules with carboxymethylcellulose (CMC) gel as a handling agent. This study investigated the potential, in vitro, of the AJBVFS to function as a delivery system for cell therapy to enhance healing of bone defects. The attachment of rabbit bone marrow stromal cells (rbBMSCs), human BMSCs (hBMSCs) and human bone-derived cells (hBDCs) to JAX granules and the effect of CMC gel on cell proliferation and differentiation were investigated. There were slight species differences in the number and morphology of cells attached on the JAX granules with less rbBMSC attachment than human. All cells tolerated the presence of CMC gel and a reduction in cell number was only seen after longer exposure to higher gel concentrations. Low concentrations of CMC gel enhanced proliferation, alkaline phosphatase (ALP) expression and ALP activity in human cells but had no effect on rbBMSC. This study suggests that AJBVFS is an appropriate scaffold for the delivery of osteogenic cells and the addition of CMC gel as a handling agent promotes osteogenic proliferation and differentiation and is therefore likely to encourage bone healing.

Effect of ternary phosphate-based glass compositions on osteoblast and osteoblast-like proliferation, differentiation and death in vitro.

There is currently a need to expand the range of graft materials available to orthopaedic surgeons. This study investigated the effect of ternary phosphate-based glass (PBG) compositions on the behaviour of osteoblast and osteoblast-like cells. PBGs of the formula (in mol.%) P(2)O(5)(50)-CaO(50-X)-Na(2)O(X), where X is either 2, 4, 6, 8 or 10, were produced and their influence on the proliferation, differentiation and death in vitro of adult human bone marrow stromal cells (hBMSCs) and human fetal osteoblast 1.19 (HFOB 1.19) cells were assessed. Tissue culture plastic (TCP) and hydroxyapatite (HA) were used as controls. Exposure to PBGs in culture inhibited cell adhesion and proliferation and increased cell death in both cell types studied. There was no significant difference in percentage cell death between the PBGs, which was significantly greater than the controls. However, compared with other PBGs, a greater number of cells were found on the 48mol.% CaO which may have been due to either increased adherence or proliferation, or both. This composition was capable of supporting osteogenic proliferation and early differentiation, and supports the notion that chemical modification of the glass could lead to a more biologically compatible substrate with the potential to support osteogenic grafting. Realisation of this potential should lead to the development of novel grafting strategies for the treatment of problematic bone defects.

Healing of an ulnar defect using a proprietary TCP bone graft substitute, JAX, in association with autologous osteogenic cells and growth factors.

Currently, available synthetic bone substitutes have adequate osteoconductive properties but have little or no osteoinductivity. Recent research has focused on using osteogenic growth factors or cells to provide this. JAX is a beta tricalcium phosphate bone graft substitute that has a novel shape and interlocking design. This study investigated delivery methods and the use of autologous cell therapy to enhance healing of a bone defect using JAX as a scaffold. Bone marrow was harvested from 24 New Zealand White rabbits. The mononuclear cell fraction was isolated and culture expanded. Bilateral 1.5 cm defects in the ulna were filled with: Group 1: JAX alone, Group 2: JAX plus 1x10(7) autologous BMSCs injected at the time of surgery, Group 3: JAX plus 8x10(6) autologous BMSCs cultured on granules for 14 days prior to surgery, Group 4: JAX plus fresh bone marrow (BMA), Group 5: cortical autograft, Group 6: JAX plus 2.5 microg VEGF. Radiographs demonstrated that there was more new bone in the BMA and VEGF groups compared to JAX alone. Groups containing autologous BMSCs were only slightly better than JAX alone in the amount of bone in the defect but did improve bridging of the osteotomy. Histomorphometry identified a significant increase in bone volume in the BMA group compared to JAX alone. BMA and VEGF enhanced healing of bone defects whereas expanded BMSCs provided little advantage over scaffold alone. There was no difference between delivery methods of autologous BMSCs. These observations suggest that the provision of osteogenic cells alone is insufficient to enhance bone healing and that additional factors are required to initiate this process in vivo.

Increased cancellous bone in the femoral neck of patients with coxarthrosis (hip osteoarthritis): a positive remodeling imbalance favoring bone formation.

Osteoporosis is caused by an imbalance between bone resorption and formation which results in an absolute reduction in bone mass. In a previous study we highlighted a condition, osteoarthritis of the hip (coxarthrosis, cOA), where an imbalance between resorption and formation provided beneficial effects in the form of an absolute increase in bone mass. We demonstrated that the femoral neck in patients with cOA had increased cancellous bone area, connectivity and trabecular thickness which might contribute to the protection against fracture associated with the condition. The aim of the present study was to analyze forming and resorbing surfaces in coxarthritic cancellous bone to assess whether increased formation or reduced resorption could be responsible for these structural changes. Whole cross-sectional femoral neck biopsies were obtained from 11 patients with cOA and histomorphometric parameters compared with 14 age- and sex-matched cadaveric controls. The ratio of osteoid surface to bone surface was 121% ( p<0.001) higher in the cases but there was no significant difference in resorptive surface. The percentage osteoid volume to bone volume (%OV/BV; +270%, p<0.001) and osteoid width (O.Wi; +127%, p<0.001) were also higher in the cases. This study suggests that the increased cancellous bone mass seen in cases of cOA is due to increased bone formation rather than decreased bone resorption. Investigation of the cellular and biochemical basis for these changes might provide new insights into the pathogenesis of osteoarthritis and highlight novel biological mechanisms regulating bone multicellular unit (BMU) balance that could be relevant to developing new interventions against hip and other osteoporotic fractures.

Increased femoral neck cancellous bone and connectivity in coxarthrosis (hip osteoarthritis).

Patients with coxarthrosis (cOA) have a reduced incidence of intracapsular femoral neck fracture, suggesting that cOA offers protection. The distribution of bone in the femoral neck was compared in cases of coxarthrosis and postmortem controls to assess the possibility that disease-associated changes might contribute to reduced fragility. Whole cross-section femoral neck biopsies were obtained from 17 patients with cOA and 22 age- and sex-matched cadaveric controls. Densitometry was performed using peripheral quantitated computed tomography (pQCT) and histomorphometry on 10-microm plastic-embedded sections. Cortical bone mass was not different between cases and controls (P > 0.23), but cancellous bone mass was increased by 75% in cOA (P = 0.014) and histomorphometric cancellous bone area by 71% (P < 0.0001). This was principally the result of an increase of apparent density (mass/vol) of cancellous bone (+45%, P = 0.001). Whereas cortical porosity was increased in the cases (P < 0.0001), trabecular width was also increased overall in the cases by 52% (P < 0.001), as was cancellous connectivity measured by strut analysis (P < 0.01). Where osteophytic bone was present (n = 9) there was a positive relationship between the amount of osteophyte and the percentage of cancellous area (P < 0.05). Since cancellous bone buttresses and stiffens the cortex so reducing the risk of buckling, the increased cancellous bone mass and connectivity seen in cases of cOA probably explain, at least in part, the ability of patients with cOA to resist intracapsular fracture of the femoral neck during a fall.

The ratio of osteocytic incorporation to bone matrix formation in femoral neck cancellous bone: an enhanced osteoblast work rate in the vicinity of hip osteoarthritis.

Recently it has been shown that an inactivating mutation in the TGFb-SMAD3 signaling pathway, which increases the conversion of osteoblasts to osteocytes, is accompanied by bone loss combined with increased osteocyte density. We hypothesized that increased matrix TGFb, known to occur in osteoarthritis, might cause the reverse of these effects in man. Because coxarthrosis (cOA) is associated with a reduced risk of femoral neck fracture, whole cross-section femoral neck biopsies were obtained from 11 patients with femoral neck fracture, 14 patients with cOA, and 22 age-and sex-matched controls. Lacunar density (Lc x mm2), osteocyte density (Ot x mm2), and cancellous wall width (Cn x W x Wi), were compared between cases of coxarthrosis, femoral neck fracture (FNF) and controls. In cOA, Lc.mm2 was reduced by 24% (P <0.001) while in FNF it was increased by 20% (P <0.001). Cn x W x Wi was increased in cOA by 22% (P <0.05) and in FNF was reduced by 27% (P <0.001). Lc x mm2 was inversely related to percentage cancellous bone area (adj. r2 = 0.373; P <0.01) and wall widths, r2 = 0.382, P <0.001. The reduction in osteocyte lacunar density coupled with increased wall width is consistent with a model of cOA effects on bone in which increased levels of matrix TGFb might prolong the effective lifespan or work rate of the osteoblast and delay its incorporation into the matrix as an osteocyte. One possible approach to strengthening bone in osteoporosis might be to enhance the effective lifespan of the osteoblast by modulating TGFb-related pathway activity in its local environment.

High concentrations of dexamethasone suppress the proliferation but not the differentiation or further maturation of human osteoblast precursors in vitro: relevance to glucocorticoid-induced osteoporosis.

OBJECTIVE: The use of glucocorticoids (GCs) in the treatment of RA is a frequent cause of bone loss. In vitro, however, this same class of steroids has been shown to promote the recruitment and/or maturation of primitive osteogenic precursors present in the colony forming unit-fibroblastic (CFU-F) fraction of human bone and marrow. In an effort to reconcile these conflicting observations, we investigated the effects of the synthetic GC dexamethasone (Dx) on parameters of growth and osteogenic differentiation in cultures of bone marrow stromal cells derived from a large cohort of adult human donors (n=30). METHODS: Marrow suspensions were cultured in the absence and presence of Dx at concentrations between 10 pm and 1 microm. After 28 days we determined the number and diameter of colonies formed, the total number of cells, the surface expression of receptors for selected growth factors and extracellular matrix proteins and, based on the expression of the developmental markers alkaline phosphatase (AP) and the antigen recognized by the STRO-1 monoclonal antibody, the proportion of cells undergoing osteogenic differentiation and their extent of maturation. RESULTS: At a physiologically equivalent concentration, Dx had no effect on the adhesion of CFU-F or on their subsequent proliferation, but did promote their osteogenic differentiation and further maturation. These effects were independent of changes in the expression of the receptors for fibroblast growth factors, insulin-like growth factor 1, nerve growth factor, platelet-derived growth factors and parathyroid hormone/parathyroid hormone-related protein, but were associated with changes in the number of cells expressing the alpha(2) and alpha(4), but not beta(1), integrin subunits. At supraphysiological concentrations, the effects of Dx on the osteogenic recruitment and maturation of CFU-F and their progeny were maintained but at the expense of a decrease in cell number. CONCLUSIONS: A decrease in the proliferation of osteogenic precursors, but not in their differentiation or maturation, is likely to be a key factor in the genesis of GC-induced bone loss.

Expression of the developmental markers STRO-1 and alkaline phosphatase in cultures of human marrow stromal cells: regulation by fibroblast growth factor (FGF)-2 and relationship to the expression of FGF receptors 1-4.

Autologous marrow stromal cells have been proposed as an adjuvant in the treatment of bone defects and diseases. This will require the development of culture conditions that permit their rapid expansion ex vivo while retaining their potential for further differentiation. Fibroblast growth factor (FGF)-2 has been proposed as a candidate for the ex vivo expansion of cells with enhanced osteogenic potential, and we have explored this possibility further using cells obtained from a large cohort of adult human donors. Treatment with FGF-2 (0.001-2.5 ng/mL) had no detectable effect on colony formation, but markedly increased their proliferative potential and that of their immediate progeny, as shown by the increases in colony size and cell number. Based on the observed increase in the expression of the developmental markers STRO-1 and alkaline phosphatase (AP), a major target for the actions of FGF-2 appears to be the more primitive cells of the osteoblast lineage, and that, when added in combination with the synthetic glucocorticoid dexamethasone (Dx), it interacts positively to promote further cell maturation. The maintenance of adequate levels of ascorbate was shown to be a critical component in determining the nature of the effect of FGF-2 on AP expression. Variation in the response (predominantly in the magnitude and/or sensitivity) of the cultured cell populations to treatment with FGF-2 was apparent, but a preliminary analysis indicated that this was not due to differences in the age or gender of the donors used. The cultured cell populations were found to express multiple FGF receptors (FGFRs; 1-4) and the observed changes in the spectrum and abundance of FGFRs expressed in relation to that of STRO-1 and AP are consistent with their expression being developmentally regulated during the process of osteogenic differentiation. These results provide novel insights into the mechanism of action of FGF-2 on human cells of the osteoblast lineage and support the use of this factor, alone or in combination with Dx, for the rapid, ex vivo expansion of cell populations with enhanced osteogenic potential.

A novel mechanism for induction of increased cortical porosity in cases of intracapsular hip fracture.

It has been suggested that, in hip fracture, the cortex on the inferoanterior (IA) to superoposterior (SP) axis is thinned and shows increased porosity. This is dependent on the presence of giant canals (i.e., diameter >385 microm), which are related to clusters of remodeling osteons. To investigate further the relationship between remodeling and bone loss, osteonal diameter (On.Dm), wall thickness (W.Th), osteoid width (O.Wi), and extent (OS) were measured in femoral neck biopsies from 12 female intracapsular hip fracture cases and 11 age- and gender-matched controls. Over 83% of giant canals were "composite" osteonal systems in which a single canal was surrounded by multiple packets of osteonal bone. Among smaller canals, over 80% of systems had a canal encircled by a single cement line containing one packet of bone ("simple"). Composites were nearly twice as prevalent in fractures (fracture cases 9.8 +/- 0.7/25 mm(2), controls 5.3 +/- 0.4/25 mm(2), p < 0. 0001), and were dependent (R(2) = 0.52) on femoral neck region (p = 0.0008) and the regional distribution of clusters of remodeling osteons (p = 0.0045). Both the inferior (I) and anterior (A) regions had an elevated number of composites (I: 263% of control values, p = 0.0054; A: 202% of control values, p = 0.0092). On.Dm was similar in fracture cases and controls (simple: fracture cases 183 +/- 3 microm, controls 191 +/- 4 microm; composites: fracture cases 446 +/- 13 microm, controls 460 +/- 13 microm). W.Th in simples was similar in fracture cases and controls (fracture cases 51 +/- 0.8 microm, controls 49 +/- 0.7 microm), but composites had significantly (p < 0. 0001) thinner walls, with the reduction in fracture cases (31%) being twice that of controls (12%, p < 0.0001). There were no differences in O.Wi. It was unusual for osteoid to fully surround the composite canal surface; OS was 38% lower in composite than simple canals (p < 0.0001). This study indicates that, in the femoral neck cortex, the principal remodeling deficit in hip fracture is specific to composite osteons. Hip fracture cases had zonal increases in composite osteon density with reduced bone formation. The data suggest that generation of composite osteons is a plausible mechanism leading to increasing porosity and trabecularization of the cortex, thus weakening the cortex in regions maximally loaded on fall impact.

Spatial clustering of remodeling osteons in the femoral neck cortex: a cause of weakness in hip fracture?

Intracapsular femoral neck fractures are associated with decreased cortical width and increased proportions of Haversian canals with diameters greater than the normal mean plus 3 SD (i.e., >385 microm). Such canals might be formed if closely associated resorbing osteons merge; a cortical event analogous with the loss of cancellous connectivity. To test this, we investigated the pattern of osteon distribution in the aging femoral neck to determine if remodeling osteons were distributed in anatomical clusters. Femoral neck biopsies from female patients with intracapsular hip fractures (n = 13) were compared with age/gender-matched cadaveric controls (n = 13). Solochrome-stained sections were analyzed for Haversian canal location, canal diameter, and the presence of an osteoid surface. Clustering was investigated using statistical software with a cluster defined as two or more osteoid-bearing osteon centers within 0.75 mm of each other. Clusters occurred more frequently than would be expected by chance (p < 0.001). Fracture cases had more clusters per unit area (3.14 +/- 0.31 clusters/25 mm2 of cortical bone) than controls (1.89 +/- 0.22) (p = 0.002). In fracture cases, the antero-inferior, antero-superior, and infero-anterior regions had more clusters per 25 mm2 than comparable control regions (ant/inf: 4.12 +/- 0.79, 1.70 +/- 0.60,p = 0.025; ant/sup: 5.31 +/- 1.1, 1.80 +/- 0.59,p = 0.013; inf/ant: 3.15 +/- 0.49, 1.27 +/-0.29, p = 0.004). The mean number of clusters per 25 mm2 per region correlated with the mean porosity per region (adjusted r2 = 0.60;p = 0.014), and the total number of giant canals per region correlated with the total number of clusters per region (adjusted r2 = 0.58; p = 0.011). In conclusion, remodeling osteons are clustered or grouped anatomically, and fracture cases have more clusters than controls. Our data suggest that merging of adjacent, clustered osteons during resorption could lead to the rapid development of canals with excessive diameters and focal weakness. Clustering is greatest in those regions that we have previously shown to have the largest relative reductions in bone strength compared with controls and known to be maximally loaded during a sideways fall. This implicates the remodeling process underlying clustering of remodeling osteons in the aetiology of hip fracture.

Further characterization of cells expressing STRO-1 in cultures of adult human bone marrow stromal cells.

Primitive cells of the osteoblast lineage are not well characterized but are known to be present within the STRO-1+ fraction of adult human bone and marrow. A survey of human osteosarcoma cell lines revealed that STRO-1 is expressed by MG-63 but not SaOS-2. Among murine cell lines tested, expression of STRO-1 was detected in the bipotential (adipocyte/osteoblast) line BMS-2 but not the committed osteoblast precursor MC3T3-E1. A proportion of cultured adult human bone marrow stromal cells (BMSCs) consistently expressed the STRO-1 antigen. The expression of a range of cell surface antigens was studied in relation to STRO-1 by flow cytometry and several, including the bone/liver/kidney isoform of alkaline phosphatase (ALP), were found to subtype the STRO-1+ population of BMSCs. Further, BMSCs dual-labeled with antibodies recognizing STRO-1 and ALP could be assigned to one of four fractions: STRO-1-/ALP-, STRO-1+/ALP-, STRO-1+/ALP+, and STRO-1-/ALP+. Cells from each fraction could be isolated in high purity and, when recultured, remained viable and exhibited a limited degree of phenotypic stability. Using reverse transcriptase-polymerase chain reaction, cells in the four fractions were found to express different levels of transcripts for the parathyroid hormone receptor (PTHr) and bone sialoprotein (BSP). The expression of transcripts for the nuclear transcription factor core-binding factor alpha 1/osteoblast-specific factor-2 (CBFA1/OSF2) was restricted to those fractions expressing STRO-1 and/or ALP. Treatment with 10 nM dexamethasone consistently increased the proportion of cells present in those fractions which expressed the highest levels of transcripts for PTHr and BSP (STRO-1+/ALP+ and STRO-1-/ALP+) while simultaneously decreasing the proportion present in the STRO-1+/ALP- fraction. In conclusion, the expression of STRO-1 in vitro remains a characteristic of less well differentiated cells of the osteoblast lineage; in cultures of BMSCs and in established human osteosarcoma cell lines, there is an inverse association between the expression of STRO-1 and ALP; dual labeling of BMSCs with monoclonal antibodies recognizing STRO-1 and ALP permits the identification and isolation of cells of the osteoblast lineage at different stages of differentiation.