Xiaoman Luo

The size of surface microstructures as an osteogenic factor in calcium phosphate ceramics

The microporosity of calcium phosphate (CaP) ceramics has been shown to have an essential role in osteoinduction by CaP ceramics after ectopic implantation. Here we show that it is not the microporosity but the size of surface microstructural features that is the most likely osteogenic factor. Two tricalcium phosphate (TCP) ceramics, namely TCP-S and TCP-B, were fabricated with equivalent chemistry and similar microporosity but different sizes of surface microstructural features. TCP-S has a grain size of 0.99 ± 0.20 μm and a micropore size of 0.65 ± 0.25 μm, while TCP-B displays a grain size of 3.08 ± 0.52 μm and a micropore size of 1.58 ± 0.65 μm. In vitro, both cell proliferation and osteogenic differentiation were significantly enhanced when human bone marrow stromal cells were cultured on TCP-S without any osteogenic growth factors, compared to TCP-B ceramic granules. The possible involvement of direct contact between cells and the TCP ceramic surface in osteogenic differentiation is also shown with a trans-well culture model. When the ceramic granules were implanted in paraspinal muscle of dogs for 12 weeks, abundant bone was formed in TCP-S (21 ± 10% bone in the available space), whereas no bone was formed in any of the TCP-B implants. The current in vitro and in vivo data reveal that the readily controllable cue, i.e. the size of the surface microstructure, could be sufficient to induce osteogenic differentiation of mesenchymal stem cells, ultimately leading to ectopic bone formation in calcium phosphate ceramics.

Zinc in calcium phosphate mediates bone induction: in vitro and in vivo model.

Zinc-containing tricalcium phosphate (Zn-TCP) was synthesized to investigate the role of zinc in osteoblastogenesis, osteoclastogenesis and in vivo bone induction in an ectopic implantation model. Zinc ions were readily released in the culture medium. Zn-TCP with the highest zinc content enhanced the alkaline phosphatase activity of human bone marrow stromal cells and tartrate-resistant acid phosphatase activity, as well as multinuclear giant cell formation of RAW264.7 monocyte/macrophages. RAW264.7 cultured with different dosages of zinc supplements in medium with or without zinc-free TCP showed that zinc could influence both the activity and the formation of multinuclear giant cells. After a 12-week implantation in the paraspinal muscle of canines, de novo bone formation and bone incidence increased with increasing zinc content in Zn-TCP - up to 52% bone in the free space. However, TCP without zinc induced no bone formation. Although the observed bone induction cannot be attributed to zinc release alone, these results indicate that zinc incorporated in TCP can modulate bone metabolism and render TCP osteoinductive, indicating to a novel way to enhance the functionality of this synthetic bone graft material.

Influence of polymer molecular weight in osteoinductive composites for bone tissue regeneration

In bone tissue regeneration, certain polymer and calcium-phosphate-based composites have been reported to enhance some biological surface phenomena, facilitating osteoinduction. Although the crucial role of inorganic fillers in heterotopic bone formation by such materials has been shown, no reports have been published on the potential effects the polymer phase may have. The present work starts from the assumption that the polymer molecular weight regulates the fluid uptake, which determines the hydrolysis rate and the occurrence of biological surface processes. Here, two composites were prepared by extruding two different molecular weight L/D,L-lactide copolymers with calcium phosphate apatite. The lower molecular weight copolymer allowed larger fluid uptake in the composite thereof, which was correlated with a higher capacity to adsorb proteins in vitro. Further, the large fluid absorption led to a quicker composite degradation that generated rougher surfaces and enhanced ion release. Following intramuscular implantation in sheep, only the composite with the lower molecular weight polymer could induce heterotopic bone formation. Besides influencing the biological potential of composites, the molecular weight also regulated their viscoelastic behaviour under cyclic stresses. The results lead to the conclusion that designing biomaterials with appropriate physico-chemical characteristics is crucial for bone tissue regeneration in mechanical load-bearing sites.

Controlling dynamic mechanical properties and degradation of composites for bone regeneration by means of filler content

Bone tissue is a dynamic composite system that adapts itself, in response to the surrounding daily (cyclic) mechanical stimuli, through an equilibrium between growth and resorption processes. When there is need of synthetic bone grafts, the biggest issue is to support bone regeneration without causing mechanically-induced bone resorption. Apart from biological properties, such degradable materials should initially support and later leave room to bone formation. Further, dynamic mechanical properties comparable to those of bone are required. In this study we prepared composites comprising calcium phosphate and L-lactide/D-lactide copolymer in various content ratios using the extrusion method. We evaluated the effect of the inorganic filler amount on the polymer phase (i.e. on the post-extrusion intrinsic viscosity). We then studied their in vitro degradation and dynamic mechanical properties (in dry and humid conditions). By increasing the filler content, we observed significant decrease of the intrinsic viscosity of the polymer phase during the extrusion process. Composites containing higher amounts of apatite had faster degradation, and were also mechanically stiffer. But, due to the lower intrinsic viscosity of their polymer phase, they had larger damping properties. Besides this, higher amounts of apatite also rendered the composites more hydrophilic letting them absorb more water and causing them the largest decrease in stiffness. These results show the importance of filler content in controlling the properties of such composites. Further, in this study we observed that the viscoelastic properties of the composite containing 50wt% apatite were comparable to those of dry human cortical bone.

Strontium-containing apatite/polylactide composites enhance bone formation in osteopenic rabbits.

UNLABELLED Strontium (Sr) has been shown to favor bone formation and is used clinically to treat osteoporosis. We have previously reported that Sr addition in apatite/polylactide composites could enhance the BMP-induced bone formation around implants at ectopic site in healthy animals. In this study we aimed to investigate the effectiveness of Sr addition on the local bone formation in osteoporosis. Apatite/polylactide composite granules with different Sr content were loaded with equal amount of rhBMP-2 and implanted intramuscularly in healthy rabbits (Con) and rabbits that received bilateral ovariectomy and daily injection of glucocorticoid (OP) for 12 weeks. The potential effect of Sr on the final volume of BMP-induced bone in both groups was investigated histologically and histomorphometrically. The de novo bone formed in OP implants was significantly less than in Con group when the implants contained no Sr, indicating that the BMP-induced osteogenesis was impaired in OP animals. Sr substitution as low as 0.5 mol% in apatite increased the bone volume in OP implants to levels comparable to that in the Con group, indicating a positive effect of Sr addition on the local bone formation in OP animals. In addition, more adipose tissue formed in parallel with the appearance of cartilage tissue in OP implants, suggesting that the differentiation potential of stem cell in OP animals may have shifted towards adipogenesis and chondrogenesis. From these results, we conclude that the use of Sr addition to enhance the bone growth surrounding implants in osteoporosis merits further study. STATEMENT OF SIGNIFICANCE The impaired bone healing capacity of osteoporotic patients might result in poor osteointegration and surgical failure in case implants are placed. In this study we aimed to enhance the bone formation around implants under such scenario by adding strontium as the stimulus. Different from other studies, the samples were loaded with rhBMP-2 and implanted at an ectopic site (spinal muscles of New Zealand rabbits) to exclude the influence of conductive bone repair. The results showed that the addition of strontium could enhance the BMP-2-induced bone formation on implants in osteopenic rabbits to levels comparable to that in healthy rabbits. Secondarily, we observed more adipose tissue and cartilage tissue in osteopenic implants, suggesting the role of adipogenesis and chondrogenesis in osteopenia/osteoporosis.

Cells responding to surface structure of calcium phosphate ceramics for bone regeneration.

Surface structure largely affects the inductive bone‐forming potential of calcium phosphate (CaP) ceramics in ectopic sites and bone regeneration in critical‐sized bone defects. Surface‐dependent osteogenic differentiation of bone marrow stromal cells (BMSCs) partially explained the improved bone‐forming ability of submicron surface structured CaP ceramics. In this study, we investigated the possible influence of surface structure on different bone‐related cells, which may potentially participate in the process of improved bone formation in CaP ceramics. Besides BMSCs, the response of human brain vascular pericytes (HBVP), C2C12 (osteogenic inducible cells), MC3T3‐E1 (osteogenic precursors), SV‐HFO (pre‐osteoblasts), MG63 (osteoblasts) and SAOS‐2 (mature osteoblasts) to the surface structure was evaluated in terms of cell proliferation, osteogenic differentiation and gene expression. The cells were cultured on tricalcium phosphate (TCP) ceramics with either micron‐scaled surface structure (TCP‐B) or submicron‐scaled surface structure (TCP‐S) for up to 14 days, followed by DNA, alkaline phosphatase (ALP) and quantitative polymerase chain reaction gene assays. HBVP were not sensitive to surface structure with respect to cell proliferation and osteogenic differentiation, but had downregulated angiogenesis‐related gene expression (i.e. vascular endothelial growth factor) on TCP‐S. Without additional osteogenic inducing factors, submicron‐scaled surface structure enhanced ALP activity and osteocalcin gene expression of human (h)BMSCs and C2C12 cells, favoured the proliferation of MC3T3‐E1, MG63 and SAOS‐2, and increased ALP activity of MC3T3‐E1 and SV‐HFO. The results herein indicate that cells with osteogenic potency (either osteogenic inducible cells or osteogenic cells) could be sensitive to surface structure and responded to osteoinductive submicron‐structured CaP ceramics in cell proliferation, ALP production or osteogenic gene expression, which favour bone regeneration. Copyright

Submicron-surface structured tricalcium phosphate ceramic enhances the bone regeneration in canine spine environment

Calcium phosphate ceramics with submicron‐scaled surface structure can trigger bone formation in non‐osseous sites and are expected to enhance bone formation in spine environment. In this study, two tricalcium phosphate ceramics having either a submicron‐scaled surface structure (TCP‐S) or a micron‐scaled one (TCP‐B) were prepared and characterized regarding their physicochemical properties. Granules (size 1–2 mm) of both materials were implanted on either left or right side of spinous process, between the two lumbar vertebrae (L3‐L4), and in paraspinal muscle of eight beagles. After 12 weeks of implantation, ectopic bone was observed in muscle in TCP‐S explants (7.7 ± 3.7%), confirming their ability to inductively form bone in non‐osseous sites. In contrast, TCP‐B implants did not lead to bone formation in muscle. Abundant bone (34.1 ± 6.6%) was formed within TCP‐S implants beside the two spinous processes, while limited bone (5.1 ± 4.5%) was seen in TCP‐B. Furthermore, the material resorption of TCP‐S was more pronounced than that of TCP‐B in both the muscle and spine environments. The results herein indicate that the submicron‐scaled surface structured tricalcium phosphate ceramic could enhance bone regeneration as compared to the micron‐scaled one in spine environment.

Topography of calcium phosphate ceramics regulates primary cilia length and TGF receptor recruitment associated with osteogenesis

Graphical abstract

Influence of fluoride in poly(d,l‐lactide)/apatite composites on bone formation

The influence of fluoride in poly(d,l-lactide)/apatite composites on ectopic bone formation was evaluated in sheep. Nano-apatite powders with different replacement levels of OH groups by fluoride (F) (0% (F0), 50% (F50), 100% (F100), and excessive (F200)) were co-extruded with poly (d,l-lactide) at a weight ratio of 1:1. Fluoride release from the composites (CF0, CF50, CF100, and CF200) was evaluated in vitro and bone formation was assessed after intramuscular implantation in sheep. After 24 weeks in simulated physiological solution, CF0 and CF50 showed negligible fluoride release, whereas it was considerable from the CF100 and CF200 composites. Histology showed that the incidence of de novo bone formation decreased in implants with increasing fluoride content indicating a negative influence of fluoride on ectopic bone formation. Furthermore, a significant decrease in resorption of the high fluoride-content composites and a reduction in the number of multinucleated giant cells were seen. These results show that instead of promoting, the presence of fluoride in poly(d,l-lactide)/apatite composites seemed to suppresses their resorption and osteoinductive potential in non-osseous sites.

1.14 Calcium Phosphates and Bone Induction

Calcium phosphates (CaPs) have a widespread use in bone repair and regeneration due to their osteoconductive and bioactive (bone bonding) nature. In the past decade, it has become apparent that a sub-group of CaPs with specific physico-chemical properties have the ability to induce bone formation in ectopic sites (ie, intramuscular or sub-cutaneously). In this chapter, we summarize the scientific evidence of CaP induced bone formation, describe material properties required for bone induction and discuss possible underlying mechanisms. In addition, we also provide data on the clinical efficacy of this unique group of CaPs.