William W. Lu

Preparation and histological evaluation of biomimetic three-dimensional hydroxyapatite/chitosan-gelatin network composite scaffolds

A novel biodegradable hydroxyapatite/chitosan-gelatin network (HA/CS-Gel) composite of similar composition to that of normal human bone was prepared as a three-dimensional biomimetic scaffold by phase separation method for bone tissue engineering. Changing the solid content and the compositional variables of the original mixtures allowed control of the porosities and densities of the scaffolds. The HA granules were dispersed uniformly in the organic network with intimate interface contact via pulverizing and ultrasonically treating commercial available HA particles. Scaffolds of 90.6% porosity were used to examine the proliferation and functions of the cells in this three-dimensional microenvironment by culturing neonatal rat caldaria osteoblasts. Histological and immunohistochemical staining and scanning electron microscopy observation indicated that the osteoblasts attached to and proliferated on the scaffolds. Extracellular matrices including collagen I and proteoglycan-like substrate were synthesized, while osteoid and bone-like tissue formed during the culture period. Furthermore, the cell/scaffold constructs had good biomineralization effect after 3 weeks in culture.

Inhibition of TGF-β signaling in mesenchymal stem cells of subchondral bone attenuates osteoarthritis

Osteoarthritis is a highly prevalent and debilitating joint disorder. There is no effective medical therapy for the condition because of limited understanding of its pathogenesis. We show that transforming growth factor β1 (TGF-β1) is activated in subchondral bone in response to altered mechanical loading in an anterior cruciate ligament transection (ACLT) mouse model of osteoarthritis. TGF-β1 concentrations are also high in subchondral bone from humans with osteoarthritis. High concentrations of TGF-β1 induced formation of nestin-positive mesenchymal stem cell (MSC) clusters, leading to formation of marrow osteoid islets accompanied by high levels of angiogenesis. We found that transgenic expression of active TGF-β1 in osteoblastic cells induced osteoarthritis, whereas inhibition of TGF-β activity in subchondral bone attenuated the degeneration of articular cartilage. In particular, knockout of the TGF-β type II receptor (TβRII) in nestin-positive MSCs led to less development of osteoarthritis relative to wild-type mice after ACLT. Thus, high concentrations of active TGF-β1 in subchondral bone seem to initiate the pathological changes of osteoarthritis, and inhibition of this process could be a potential therapeutic approach to treating this disease.Osteoarthritis is a highly prevalent and debilitating joint disorder. There is no effective medical therapy for osteoarthritis due to limited understanding of osteoarthritis pathogenesis. We show that TGF–β1 is activated in the subchondral bone in response to altered mechanical loading in an anterior cruciate ligament transection (ACLT) osteoarthritis mouse model. TGF–β1 concentrations also increased in human osteoarthritis subchondral bone. High concentrations of TGF–β1 induced formation of nestin+ mesenchymal stem cell (MSC) clusters leading to aberrant bone formation accompanied by increased angiogenesis. Transgenic expression of active TGF–β1 in osteoblastic cells induced osteoarthritis. Inhibition of TGF–β activity in subchondral bone attenuated degeneration of osteoarthritis articular cartilage. Notably, knockout of the TGF–β type II receptor (TβRII) in nestin+ MSCs reduced development of osteoarthritis in ACLT mice. Thus, high concentrations of active TGF–β1 in the subchondral bone initiated the pathological changes of osteoarthritis, inhibition of which could be a potential therapeutic approach.

Three-dimensional nanocomposite scaffolds fabricated via selective laser sintering for bone tissue engineering.

Bionanocomposites formed by combining biodegradable polymers and nanosized osteoconductive inorganic solids have been regarded as promising biomimetic systems which possess much improved structural and functional properties for bone tissue regeneration. In this study three-dimensional nanocomposite scaffolds based on calcium phosphate (Ca-P)/poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and carbonated hydroxyapatite (CHAp)/poly(l-lactic acid) (PLLA) nanocomposite microspheres were successfully fabricated using selective laser sintering, which is a rapid prototyping technology. The sintered scaffolds had controlled material microstructure, totally interconnected porous structure and high porosity. The morphology and mechanical properties of Ca-P/PHBV and CHAp/PLLA nanocomposite scaffolds as well as PHBV and PLLA polymer scaffolds were studied. In vitro biological evaluation showed that SaOS-2 cells had high cell viability and normal morphology and phenotype after 3 and 7 days culture on all scaffolds. The incorporation of Ca-P nanoparticles significantly improved cell proliferation and alkaline phosphatase activity for Ca-P/PHBV scaffolds, whereas CHAp/PLLA nanocomposite scaffolds exhibited a similar level of cell response compared with PLLA polymer scaffolds. The nanocomposite scaffolds provide a biomimetic environment for osteoblastic cell attachment, proliferation and differentiation and have great potential for bone tissue engineering applications.

Effects of strontium in modified biomaterials

Strontium (Sr) plays a special role in bone remodelling, being associated with both the stimulation of bone formation and a reduction in bone resorption. Thus, the modification of biomaterials by partial or full substitution by Sr is expected to increase both bioactivity and biocompatibility. However, such effects have to be studied individually. Although no phase transition was found in Sr-substituted hydroxyapatite (Sr-HA), Sr-containing calcium silicate (Sr-CS) or Sr-containing borosilicate (Sr-BS), their biological performance was substantially affected by changes in the physico-chemical properties and Sr content of the materials. Three distinct outcomes were found for the presence of Sr: (1) increased HA solubility; (2) no significant effect on the degradation rate of CS; (3) apparent inhibition of the otherwise rapid degradation of BS. In each case the released Sr affected osteoblast proliferation and alkaline phosphatase activity, with clear evidence that an optimum Sr dose exists. Such chemical and biological variations must be disentangled for the behaviour to be properly understood and materials design to be advanced.

Strontium Promotes Osteogenic Differentiation of Mesenchymal Stem Cells Through the Ras/MAPK Signaling Pathway

Strontium ralenate is a new anti-osteoporosis agent. The cellular and molecular mechanism underlying the anabolic effect of strontium on bone remains to be elucidated. Osteoblasts, the main bone forming cells are known to be derived from bone marrow mesenchymal stem cells (MSCs). The present study therefore aimed to investigate the possible effects of strontium on MSCs and signaling pathways possibly involved. It was firstly demonstrated that strontium treatment significantly increased osteoblast-related gene expression and alkaline phosphatase (ALP) of osteogenic-differentiating MSCs. Accompanying the enhanced osteogenic differentiation, the increased phosphorylation of mitogen-activated protein kinase (MAPK) ERK1/2 and p38 was detected in strontium-treated MSCs. PD98059 and SB203580, selective inhibitors of ERK1/2 kinase and p38, attenuated the effect of strontium on osteogenesis. Furthermore, it was demonstrated that Rat Sarcoma viral oncogene homolog (RAS), an upstream regulator of ERK1/2 and p38, was activated by strontium treatment and siRNA-mediated Ras knockdown inhibited strontium-stimulated expression of osteogenic markers. Finally, the transcriptional activity and phosphorylation level of Runx2 was significantly increased in response to strontium treatment in MSCs. PD98059 and Ras siRNA inhibited the effect of strontium on Runx2 activation. Taken together, these results indicated that strontium can promote osteogenic differentiation of MSCs through activating the Ras/MAPK signaling pathway and the downstream transcription factor Runx2.

Palmar Plate Fixation of AO Type C2 Fracture of Distal Radius Using a Locking Compression Plate –A Biomechanical Study in a Cadaveric Model

The stability of palmar plate fixation using a locking compression T-plate was compared with that of a conventional palmar T-plate and a dorsal T-plate in a cadaveric model of an AO type C2 fracture of distal radius. The wrist axial load transmission through the radius was tested for each fixation. The results show that, under 100N axial load, the palmar locking compression T-plate restores stability comparable to that of the intact radius, and is superior to conventional palmar or dorsal T-plates.

Enhanced osteoporotic bone regeneration by strontium-substituted calcium silicate bioactive ceramics

The regeneration capacity of the osteoporotic bones is generally lower than that of the normal bones. Current methods of bone defect treatment for osteoporosis are not always satisfactory. Recent studies have shown that the silicate based biomaterials can stimulate osteogenesis and angiogenesis due to the silicon (Si) ions released from the materials, and enhance bone regeneration in vivo. Other studies showed that strontium (Sr) plays a distinct role on inhibiting bone resorption. Based on the hypothesis that the combination of Si and Sr may have synergetic effects on osteoporotic bone regeneration, the porous Sr-substituted calcium silicate (SrCS) ceramic scaffolds combining the functions of Sr and Si elements were developed with the goals to promote osteoporotic bone defect repair. The effects of the ionic extract from SrCS on osteogenic differentiation of bone marrow mesenchymal stem cells derived from ovariectomized rats (rBMSCs-OVX), angiogenic differentiation of human umbilical vein endothelial cells (HUVECs) were investigated. The in vitro results showed that Sr and Si ions released from SrCS enhanced cell viability, alkaline phosphatase (ALP) activity, and mRNA expression levels of osteoblast-related genes of rBMSCs-OVX and expression of vascular endothelial growth factor (VEGF) without addition of extra osteogenic and angiogenic reagents. The activation in extracellular signal-related kinases (ERK) and p38 signaling pathways were observed in rBMSCs-OVX cultured in the extract of SrCS, and these effects could be blocked by ERK inhibitor PD98059, and P38 inhibitor SB203580, respectively. Furthermore, the ionic extract of SrCS stimulated HUVECs proliferation, differentiation and angiogenesis process. The in vivo experiments revealed that SrCS dramatically stimulated bone regeneration and angiogenesis in a critical sized OVX calvarial defect model, and the enhanced bone regeneration might be attributed to the modulation of osteogenic differentiation of endogenous mesenchymal stem cells (MSCs) and the inhibition of osteoclastogenesis, accompanying with the promotion of the angiogenic activity of endothelial cells (ECs).

Apatite-formation ability--predictor of "bioactivity"?

The ability to trigger the formation of apatite from a supersaturated solution has been widely used to imply the bioactivity of an implant in vivo. However, the method itself may provide at best incomplete information, primarily because it is determined only by solution supersaturation, irrespective of biological processes. Bone regeneration is triggered mainly by the vitality of osteoblasts, and regulated by the expression of growth factors such as oestrogen, parathyroid hormone and bone morphogenetic proteins, while ions or other species released from an implant may affect the expression of such growth factors, and so bone resorption or formation. The misinterpretation of the outcome of such tests must result in misunderstanding of the true effects and behaviour of materials intended for use in embedded biological contexts. Thus, the underlying and motivating hypothesis needs to be carefully reconsidered, along with the results of all work founded on the concept. It would seem that it is only viable to test using osteoblasts, whether in vivo or in vitro.

Irradiation induces bone injury by damaging bone marrow microenvironment for stem cells

Radiation therapy can result in bone injury with the development of fractures and often can lead to delayed and nonunion of bone. There is no prevention or treatment for irradiation-induced bone injury. We irradiated the distal half of the mouse left femur to study the mechanism of irradiation-induced bone injury and found that no mesenchymal stem cells (MSCs) were detected in irradiated distal femora or nonirradiated proximal femora. The MSCs in the circulation doubled at 1 week and increased fourfold after 4 wk of irradiation. The number of MSCs in the proximal femur quickly recovered, but no recovery was observed in the distal femur. The levels of free radicals were increased threefold at 1 wk and remained at this high level for 4 wk in distal femora, whereas the levels were increased at 1 wk and returned to the basal level at 4 wk in nonirradiated proximal femur. Free radicals diffuse ipsilaterally to the proximal femur through bone medullary canal. The blood vessels in the distal femora were destroyed in angiographic images, but not in the proximal femora. The osteoclasts and osteoblasts were decreased in the distal femora after irradiation, but no changes were observed in the proximal femora. The total bone volumes were not affected in proximal and distal femora. Our data indicate that irradiation produces free radicals that adversely affect the survival of MSCs in both distal and proximal femora. Irradiation injury to the vasculatures and the microenvironment affect the niches for stem cells during the recovery period.

Inhibition of Sca-1-Positive Skeletal Stem Cell Recruitment by Alendronate Blunts the Anabolic Effects of Parathyroid Hormone on Bone Remodeling

The anabolic effects of parathyroid hormone (PTH) on bone formation are impaired by concurrent use of antiresorptive drugs. We found that the release of active transforming growth factor (TGF)-β1 during osteoclastic bone resorption is inhibited by alendronate. We showed that mouse Sca-1-positive (Sca-1(+)) bone marrow stromal cells are a skeletal stem cell subset, which are recruited to bone remodeling sites by active TGF-β1 in response to bone resorption. Alendronate inhibits the release of active TGF-β1 and the recruitment of Sca-1(+) skeletal stem cells for the bone formation. The observation was validated in a Tgfb1(-/-) mouse model, in which the anabolic effects of PTH on bone formation are diminished. The PTH-stimulated recruitment of injected mouse Sca-1(+) cells to the resorptive sites was inhibited by alendronate. Thus, inhibition of active TGF-β1 release by alendronate reduces the recruitment of Sca-1(+) skeletal stem cells and impairs the anabolic action of PTH in bone.