Karin A. Hing

A comparative study on the in vivo behavior of hydroxyapatite and silicon substituted hydroxyapatite granules

Phase pure hydroxyapatite (HA) and a 0.8 wt % silicon substituted hydroxyapatite (SiHA) were prepared by aqueous precipitation methods. Both HA and SiHA were processed into granules 0.5–1.0 mm in diameter and sintered at 1200 °C for 2 h. The sintered granules underwent full structural characterization, prior to implantation into the femoral condyle of New Zealand White rabbits for a period of 23 days. The results show that both the HA and SiHA granules were well accepted by the host tissue, with no presence of any inflammatory cells. New bone formation was observed directly on the surfaces and in the spaces between both HA and SiHA granular implants. The quantitative histomorphometry results indicate that the percentage of bone ingrowth for SiHA (37.5%±5.9) was significantly greater than that for phase pure HA (22.0%±6.5), in addition the percentage of bone/implant coverage was significantly greater for SiHA (59.8%±7.3) compared to HA (47.1%±3.6). These findings indicate that the early in vivo bioactivity of hydroxyapatite was significantly improved with the incorporation of silicate ions into the HA structure, making SiHA an attractive alternative to conventional HA materials for use as bone substitute ceramics.

CHARACTERIZATION OF POROUS HYDROXYAPATITE

Hydroxyapatite has been considered for use in the repair of osseous defects for the last 20 years. Recent developments have led to interest in the potential of porous hydroxyapatite as a synthetic bone graft. However, despite considerable activity in this field, regarding assessment of the biological response to such materials, the basic materials characterization is often inadequate. This paper documents the characterization of the chemical composition, mechanical integrity, macro- and microstructure of a porous hydroxyapatite, Endobon ® (E. Merck GmbH), intended for the bone-graft market. Specimens possesed a range of apparent densities from 0.35 to 1.44 g cm-3. Chemical analysis demonstrated that the natural apatite precursor of Endobon® was not converted to pure hydroxyapatite, but retained many of the ionic substituents found in bone mineral, notably carbonate, sodium and magnesium ions. Investigation of the microstructure illustrated that the struts of the material were not fully dense, but had retained some traces of the network of osteocyte lacunae. Macrostructural analysis demonstrated the complex inter-relationship between the structural features of an open pore structure. Both pore size and connectivity were found to be inversely dependent on apparent density. Furthermore, measurement of pore aspect ratio and orientation demonstrated a relationship between apparent density and the degree of macrostructural anisotropy within the specimens, while, it was also noted that pore connectivity was sensitive to anisotropy. Compression testing demonstrated the effect of apparent density and macrostructural anisotropy on the mechanical properties. An increase in apparent density from 0.38 to 1.25 g cm-3 resulted in increases in ultimate compressive stress and compressive modulus of 1 to 11 MPa and 0.2 to 3.1 GPa, respectively. Furthermore, anisotropic high density (> 0.9 g cm-3) specimens were found to possess lower compressive moduli than isotropic specimens with equivalent apparent densities. These results underline the importance of full structural and mechanical characterization of porous ceramic implant materials. ©1999 Kluwer Academic Publishers

Quantification of bone ingrowth within bone-derived porous hydroxyapatite implants of varying density

Hydroxyapatite has been investigated for use in the osseous environment for over 20 years and the biocompatibility of the ceramic and its osseoconductive behavior is well established. Therefore, the use of porous hydroxyapatite for the repair of osseous defects seems promising with potential for complete penetration of osseous tissue and restoration of vascularity throughout the repair site. However, there have been few systematic studies of the effects of physical properties such as macropore size and pore connectivity on the rate and quality of bone integration within porous hydroxyapatite implants. This paper quantifies the early biological response to a well-characterized series of implants with uniform microstructure and phase composition, but differing macrostructures and demonstrates the dependence of the rate of osseointegration on the apparent density of porous hydroxyapatite as a function of pore connectivity. Furthermore, compression testing established that bony ingrowth has a strong reinforcing effect on porous hydroxyapatite implants, which is more pronounced in the lower density implants, as a result of a greater relative volume of bone ingrowth.

Porosity variation in hydroxyapatite and osteoblast morphology: a scanning electron microscopy study

The biocompatibility of hydroxyapatite has been demonstrated by previous studies, with enhancement of osteointegration through the use of porous hydroxyapatite (pHA). Emphasis has been focused on the use of coralline hydroxyapatite or the introduction of macroporosity into synthetic hydroxyapatite. The current study investigates the role of macro‐ and microporosities in synthetic phase‐pure porous hydroxyapatite on the morphological aspects of human osteoblast‐like cells using scanning electron microscopy. Cells were seeded on four different types of porous hydroxyapatite (HA1, HA2, HA3 and HA4) and examined following 1, 2, 14 and 30 days of incubation in vitro. The results indicated that the cells had an affinity to micropores through filopodia extensions, at initial stage of attachment. Cellular proliferation and colonization was evident on all materials with cells forming cellular bridges across the macropores at day 14 with cellular canopy formation covering entire macropores observed by day 30. This study demonstrates that while the introduction of microporosity has no evident effect on cellular morphology at later time points, it seems to play a role in initial cellular anchorage and attachment.

The effects of microporosity on osteoinduction of calcium phosphate bone graft substitute biomaterials.

The effect of increasing strut porosity on the osteoinductive ability of silicate substituted calcium phosphate (SiCaP) biomaterials was investigated in an ectopic ovine model. Implants with strut porosities of 22.5%, 32.0% and 46.0% were inserted into the parapsinalis muscle. At 8, 12 and 24 weeks histological sections were prepared. Sections were examined using backscattered scanning electron microscopy and un-decalcified histology. Bone area, implant area and bone-implant contact were quantified. At 8 weeks there was no significant difference between the groups in terms of bone area and implant area. However at 12 weeks, the amount of bone formation observed was significantly greater in SiCaP-46 (6.17 ± 1.51%) when compared with SiCaP-22.5 (1.33 ± 0.84%) p=0.035. Results also showed significantly increased amounts of bone-implant contact to the SiCaP-46 scaffold (3.30 ± 1.17%) compared with SiCaP-22.5 (0.67 ± 0.52%, p=0.043) at 8 weeks and 12 weeks; (SiCaP-46 (21.82 ± 5.59%) vs SiCaP-22.5 (3.06 ± 1.89%), p=0.012). At 24 weeks, bone formation and graft resorption had significantly increased in all groups so that the level of bone formation in the SiCaP-46 group had increased 75-fold to 30.05 ± 8.38%. Bone formation was observed in pores <10 μm. Results suggest that bone graft substitute materials with greater strut porosity are more osteoinductive.

Biomechanical assessment of bone ingrowth in porous hydroxyapatite

Porous hydroxyapatite (Endobon®) specimens were implanted into the femoral condyle of New Zealand White rabbits for up to 6 months. After sacrifice, specimens were sectioned for histology and mechanical testing, where the extent of reinforcement by bony ingrowth was assessed by compression testing and fixation was assessed by push-out testing. From histological observations, it was established that the majority of bone ingrowth occurred between 10 day and 5 weeks after implantation and proceeded predominantly from the deep end of the trephined defect, with some integration from the circumferential sides. At 3 months, the implants were fully integrated, exhibiting bony ingrowth, vascularization and bone marrow stroma within the internal macropores. After 5 weeks, the mean ultimate compressive strength of retrieved implants (6.9 MPa) was found to be greater than that of the original implant (2.2 MPa), and by 3 months the fully integrated implants attained a compressive strength of approximately 20 MPa. Push-out testing demonstrated that after 5 weeks in vivo, the interfacial shear strength reached 3.2 MPa, increasing to 7.3 MPa at 3 and 6 months.

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.

Hydroxyapatite promotes superior keratocyte adhesion and proliferation in comparison with current keratoprosthesis skirt materials

Aim: Published clinical series suggest the osteoodontokeratoprosthesis (OOKP) may have a lower extrusion rate than current synthetic keratoprostheses. The OOKP is anchored in the eye wall by autologous tooth. The authors’ aim was to compare adhesion, proliferation, and morphology for telomerase transformed keratocytes seeded on calcium hydroxyapatite (the principal mineral constituent of tooth) and materials used in the anchoring elements of commercially available synthetic keratoprostheses. Methods: Test materials were hydroxyapatite, polytetrafluoroethylene (PTFE), polyhydroxyethyl methacrylate (HEMA), and glass (control). Cell adhesion and viability were quantified at 4 hours, 24 hours, and 1 week using a calcein-AM/EthD-1 viability/cytotoxicity assay. Focal contact expression and cytoskeletal organisation were studied at 24 hours by confocal microscopy with immunoflourescent labelling. Further studies of cell morphology were performed using light and scanning electron microscopy. Results: Live cell counts were significantly greater on hydroxyapatite surfaces at each time point (p<0.04). Dead cell counts were significantly higher for PTFE at 7 days (p<0.002). ß1 integrin expression was highest on hydroxyapatite. Adhesion structures were well expressed in flat, spread out keratocytes on both HA and glass. Keratocytes tended to be thinner and spindle shaped on PTFE. The relatively few keratocytes visible on HEMA test surfaces were rounded and poorly adherent. Conclusions: Keratocyte adhesion, spreading, and viability on hydroxyapatite test surfaces is superior to that seen on PTFE and HEMA. Improving the initial cell adhesion environment in the skirt element of keratoprostheses may enhance tissue integration and reduce device failure rates.

Histomorphological and biomechanical characterization of calcium phosphates in the osseous environment

Abstract The standardization of characterization techniques is becoming increasingly important for bone replacement materials as it becomes apparent that, for the field to advance, testing must be developed to allow the biocompatibility or bioactivity of a new material to be assessed and directly compared with existing materials. Currently there are many forms of biocompatibility test for materials destined for the osseous environment, ranging from immersion in simulated body fluid to implantation into living bone. However, the variety of ways in which the data from these tests may be acquired and interpreted, as a result of changes in parameters such as surgical technique and mechanical test conditions, means that much of the published data within the field is not comparable. This paper will introduce the concept of biocompatibility by considering calcium phosphate bioceramics, and discusses some aspects of in vivo experimental design, including simple histomorphometry techniques, in addition to considering practical methods for the assessment of the biomechanical characteristics of an osseointegrated implant.

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.