A carboxymethyl cellulose bone graft carrier delays early bone healing in an ovine model.

A limitation in the use of calcium phosphate (CaP) is that in its raw form, it comprises blocks or granules, which are limited in their utility for orthopedic surgery and a number of commercial bone grafts are supplied within an aqueous based carboxymethyl cellulose (CMC) putty. Our hypothesis was that CMC combined with a porous silicate-substituted CaP (SiCaP) scaffold would have no negative effect on bone formation after implantation in an ovine femoral condyle. Defects were either (a) empty or filled with (b) SiCaP granules, (c) CMC-SiCaP Putty or (d) a SiCaP press-fit dry block. Scaffolds were identical in composition and remained in vivo for 4, 8, and 12 weeks. Bone apposition rates, bone area, percentage of bone-implant contact and graft area were quantified. At 4 and 8 weeks, significantly more new bone and percentage of bone-implant contact was measured within granules when compared with both putty and block scaffolds. At 12 weeks, significantly increased bone was measured for the granules when compared with blocks and no significant difference was found when the granules and putty scaffolds were compared. Results showed the disadvantageous effect that CMC may have on early bone growth and that granules increased new bone formation when compared with a press-fit block composed of the same material.

The effect of increased microporosity on bone formation within silicate-substituted scaffolds in an ovine posterolateral spinal fusion model.

This study compared the bone forming capacity of the same formulation of silicate-substituted bone graft substitute materials with different microporosity in an instrumented posterolateral spinal fusion ovine model. Materials with a strut porosity of (i) 22.5% (SiCaP) or (ii) 36.0% (SiCaP(+)) were packed along either side of the spine. Bone apposition rates, % new bone formation, % bone-implant contact, and % graft resorption were quantified at 8, 12, and 24 weeks post surgery. Computed Tomography (CT) was used to grade the formation of fusion bridges between vertebrae. Results showed no significant difference in bone apposition rates, % new bone formation, and % bone-implant contact when the two materials were compared. However, at 8 weeks, a significantly higher CT score was obtained in the SiCaP(+) group (0.83 ± 0.17) when compared with the SiCaP group (0.17 ± 0.17; p = 0.027). Significantly less scaffold remained in the SiCaP(+) group at 12 weeks (p = 0.018). Both SiCaP and SiCaP(+) formulations augmented bone formation. Increasing the strut porosity did not significantly increase bone formation however, at 8 weeks it promoted the formation of more highly mineralized bone resulting in a significantly higher CT score, suggesting the bone tissue formed was more mature. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 805-814, 2017.

Efficacy of silicate-substituted calcium phosphate with enhanced strut porosity as a standalone bone graft substitute and autograft extender in an ovine distal femoral critical defect model.

A synthetic bone graft substitute consisting of silicate-substituted calcium phosphate with increased strut porosity (SiCaP EP) was evaluated in an ovine distal femoral critical sized metaphyseal defect as a standalone bone graft, as an autologous iliac crest bone graft (ICBG) extender (SiCaP EP/ICBG), and when mixed with bone marrow aspirate (SiCaP EP/BMA). Defects were evaluated after 4, 8, and 12 weeks with radiography, decalcified paraffin-embedded histopathology, non-decalcified resin-embedded histomorphometry, and mechanical indentation testing. All test groups exhibited excellent biocompatibility and osseous healing as evidenced by an initial mild inflammatory response followed by neovascularization, bone growth, and marrow infiltration throughout all SiCaP EP-treated defects. SiCaP EP/ICBG produced more bone at early time points, while all groups produced similar amounts of bone at later time points. SiCaP EP/ICBG likewise showed more favorable mechanical properties at early time points, but was equivalent to SiCaP EP and SiCaP EP/BMA at later time points. This study demonstrates that SiCaP EP is efficacious as a standalone bone graft substitute, mixed with BMA, and as an autograft extender.

Silicate-substituted calcium phosphate with enhanced strut porosity stimulates osteogenic differentiation of human mesenchymal stem cells.

While many synthetic ceramic bone graft substitutes (BGSs) have osteoconductive properties (e.g. provide a physical scaffold for osteointegration of surrounding bone tissue), certain BGSs are osteostimulative in that they actively upregulate mesenchymal stem cell proliferation and stimulate differentiation into osteoblast-like cells. The osteostimulative properties of silicate-substituted calcium phosphate with enhanced porosity (SiCaP EP) were evaluated in vitro with STRO-1+ immunoselected human bone marrow derived mesenchymal stem cells (HBMSCs). Osteostimulative materials (SiCaP) and Bioglass 45S5 (Bioglass) were also assessed as positive controls along with non-silicate substituted hydroxyapatite as a negative control. HBMSCs were also assessed on Thermanox discs cultured in basal and osteogenic media to determine when osteogenic differentiation could be significantly detected with this in vitro cell system. HBMSC viability and necrosis, total DNA content, alkaline phosphatase (ALP) expression, and osteocalcin expression were evaluated after 7, 14, 21, and 28 days. It was demonstrated that SiCaP EP is osteostimulative based on its propensity to support STRO-1+ HBMSC proliferation and ability to promote the differentiation of HBMSCs down the osteoblastic lineage from ALP-expressing, matrix-producing osteoblasts to Osteocalcin-producing pre-osteocytes without the presence of external osteogenic factors. SiCaP EP permitted greater HBMSC attachment as well as ALP and Osteocalcin expression than Bioglass which may be attributed to its microstructure and chemistry.

The effect of particle size on the osteointegration of injectable silicate-substituted calcium phosphate bone substitute materials.

Calcium phosphate (CaP) particles as a carrier in an injectable bone filler allows less invasive treatment of bony defects. The effect of changing granule size within a poloxamer filler on the osteointegration of silicate-substituted calcium phosphate (SiCaP) bone substitute materials was investigated in an ovine critical-sized femoral condyle defect model. Treatment group (TG) 1 consisted of SiCaP granules sized 1000-2000 µm in diameter (100 vol %). TG2 investigated a granule size of 250-500 µm (75 vol %), TG3 a granule size of 90-125 µm (75 vol %) and TG4 a granule size of 90-125 µm (50 vol %). Following a 4 and 8 week in vivo period, bone area, bone-implant contact, and remaining implant area were quantified within each defect. At 4 weeks, significantly increased bone formation was measured in TG2 (13.32% ± 1.38%) when compared with all other groups (p = 0.021 in all cases). Bone in contact with the bone substitute surface was also significantly higher in TG2. At 8 weeks most new bone was associated within defects containing the smallest granule size investigated (at the lower volume) (TG4) (42.78 ± 3.36%) however this group was also associated with higher amounts of fragmented SiCaP. These smaller particles were phagocytosed by macrophages and did not appear to have a negative influence on healing. In conclusion, SiCaP granules of 250-500 µm in size may be a more suitable scaffold when used as an injectable bone filler and may be a convenient method for treating bony defects.

Directed osteogenic differentiation of human mesenchymal stem/precursor cells on silicate substituted calcium phosphate.

Insufficient, underactive, or inappropriate osteoblast function results in serious clinical conditions such as osteoporosis, osteogenesis imperfecta and fracture nonunion and therefore the control of osteogenesis is a medical priority. In vitro mesenchymal stem cells (MSCs) can be directed to form osteoblasts through the addition of soluble factors such as ß-glycerophosphate, ascorbic acid, and dexamethasone; however this is unlikely to be practical in the clinical setting. An alternative approach would be to use a scaffold or matrix engineered to provide cues for differentiation without the need for soluble factors. Here we describe studies using Silicate-substituted calcium phosphate (Si-CaP) and unmodified hydroxyapatite (HA) to test whether these materials are capable of promoting osteogenic differentiation of MSCs in the absence of soluble factors. Si-CaP supported attachment and proliferation of MSCs and induced osteogenesis to a greater extent than HA, as evidenced through upregulation of the osteoblast-related genes: Runx2 (1.2 fold), Col1a1 (2 fold), Pth1r (1.5 fold), and Bglap (1.7 fold) Dmp1 (1.1 fold), respectively. Osteogenic-associated proteins, alkaline phosphatase (1.4 fold), RUNX2, COL1A1, and BGLAP, were also upregulated and there was an increased production of mineralized bone matrix (1.75 fold), as detected by the Von Kossa Assay. These data indicate that inorganic substrates are capable of directing the differentiation programme of stem cells in the absence of known chemical drivers and therefore may provide the basis for bone repair in the clinical setting.

Microstructure and chemistry affects apatite nucleation on calcium phosphate bone graft substitutes.

The bioactivity of calcium phosphate bone grafts of varying chemistry and strut-porosity was compared by determining the rate of formation of hydroxycarbonate apatite crystals on the material surface after being soaked in simulated body fluid for up to 30 days. Three groups of silicate-substituted hydroxyapatite material were tested, with each group comprising a different quantity of strut-porosity (23, 32, and 46 % volume). A commercially available porous ß-tricalcium phosphate bone graft substitute was tested for comparison. Results indicate that strut-porosity of a material affects the potential for formation of a precursor to bone-like apatite and further confirms previous findings that ß-tricalcium phosphate is less bioactive than hydroxyapatite.

Effect of increased strut porosity of calcium phosphate bone graft substitute biomaterials on osteoinduction.

The effect of increasing strut porosity on the osteoinductivity of porous calcium phosphate (CaP) and silicate-substituted calcium phosphate (SiCaP) bone substitute materials was investigated in an ovine ectopic model. One to two millimeter-sized granules or block implants with strut porosities of 10, 20, or 30% were inserted into the left and right paraspinalis muscle. At 12 weeks, histological sections were prepared through the center of each implant and bone contact, bone area and implant area quantified. Backscattered scanning electron microscopy (bSEM) was used to visualize bone within small pores in the struts of the scaffolds. Increased bone formation was measured in the SiCaP with 30% strut porosity (5.482% ± 1.546%) when compared with the nonsilicate CaP with the same morphology (1.160% ± 0.502%, p = 0.02), indicating that silicate substitution may increase osteoinduction. Greater bone formation was seen in scaffolds with increased strut porosity. No bone growth was found in any of the SiCaP scaffold with 10% porosity. There was no significant difference between block and granule specimens. Scanning electron microscopy and EDX in combination with histology demonstrated bone formation within pores <5 µm in size. The use of silicate-substituted CaP material with increased strut porosity may further augment repair and regeneration in bony sites.

Effects of serum protein on ionic exchange between culture medium and microporous hydroxyapatite and silicate-substituted hydroxyapatite.

It has been proposed that one of the underlying mechanisms contributing to the bioactivity of osteoinductive or osteoconductive calcium phosphates involves the rapid dissolution and net release of calcium and phosphate ions from the matrix as alternatively a precursor to subsequent re-precipitation of a bone-like apatite at the surface and/or to facilitate ion exchange in biochemical processes. In order to confirm and evaluate ion release from sintered hydroxyapatite (HA) and to examine the effect of silicate substitution into the HA lattice on ion exchange under physiological conditions we monitored Ca(2+), PO(4)(3-) and SiO(4)(4-) levels in Earl's minimum essential medium (E-MEM) in the absence (serum-free medium, SFM) or presence (complete medium, C-MEM) of foetal calf serum (FCM), with both microporous HA or 2.6 wt% silicate-substituted HA (SA) sintered discs under both static and semi-dynamic (SD) conditions for up to 28 days. In SFM, variation in Ca(2+) ion concentration was not observed with either disc chemistry or culture conditions. In C-MEM, Ca(2+) ions were released from SA under static and SD conditions whereas with HA Ca(2+) was depleted under SD conditions. PO(4)(3-) depletion occurred in all cases, although it was greater in C-MEM, particularly under SD conditions. SiO(4)(4-) release occurred from SA irrespective of medium or culture conditions but a sustained release only occurred in C-MEM under SD conditions. In conclusion we showed that under physiological conditions the reservoir of exchangeable ions in both HA and SA in the absence of serum proteins is limited, but that the presence of serum proteins facilitated greater ionic exchange, particularly with SA. These observations support the hypothesis that silicate substitution into the HA lattice facilitates a number of ionic interactions between the material and the surrounding physiological environment, including but not limited to silicate ion release, which may play a key role in determining the overall bioactivity and osteoconductivity of the material. However, significant net release of Ca(2+) and PO(4)(3-) was not observed, thus rapid or significant net dissolution of the material is not necessarily a prerequisite for bioactivity in these materials.

Increasing strut porosity in silicate-substituted calcium-phosphate bone graft substitutes enhances osteogenesis.

Synthetic, porous silicate-substituted calcium phosphate bone graft matrices (SiCaP; 0.8 wt % Si) with varying strut porosity were applied to ovine critical-sized defect sites as either 1-2 mm microgranules (SiCaP-23G, SiCaP-32G, and SiCaP-46G) or 1-2 mm microgranules in an aqueous poloxamer carrier (SiCaP-23P, SiCaP-32P, and SiCaP-46P). Defect sites treated with SiCaP-23G or SiCaP-23P showed evidence of bone formation at 8 and 12 weeks in central zones. More advanced neovascularization and increased bone contact was observed for graft materials with higher strut porosities. At 12 weeks, graft materials with higher strut porosities (32% and 46%) had statistically significantly higher absolute bone volumes (p < 0.05) versus those with a strut porosity of 23%. Absolute bone volume in defects treated with grafts of matched strut porosities as microgranules, or microgranules with poloxamer carrier, were similar at 12 weeks. Absolute graft volume for SiCaP-46 reduced over 12 weeks (not statistically significant). In conclusion, bone formation patterns in critically-sized defects confirm strut porosity to be a clinically relevant property of porous silicate-substituted calcium phosphate bone grafts in promoting osteogenesis. Increasing graft matrix strut porosity encouraged earlier neovascularization and increased the absolute equilibrium volume of bone growth within the graft without compromising graft stability.