Graphene-modified CePO4 nanorods effectively treat breast cancer-induced bone metastases and regulate macrophage polarization to improve osteo-inductive ability.

BACKGROUND: Breast cancer bone metastasis has become one of the most common complications; however, it may cause cancer recurrence and bone nonunion, as well as local bone defects. METHODS: Herein, In vitro, we verified the effect of bioscaffold materials on cell proliferation and apoptosis through a CCK8 trial, staining of live/dead cells, and flow cytometry. We used immunofluorescence technology and flow cytometry to verify whether bioscaffold materials regulate macrophage polarization, and we used ALP staining, alizarin red staining and PCR to verify whether bioscaffold material promotes bone regeneration. In vivo, we once again studied the effect of bioscaffold materials on tumors by measuring tumor volume in mice, Tunel staining, and caspase-3 immunofluorescence. We also constructed a mouse skull ultimate defect model to verify the effect on bone regeneration. RESULTS: Graphene oxide (GO) nanoparticles, hydrated CePO4 nanorods and bioactive chitosan (CS) are combined to form a bioactive multifunctional CePO4/CS/GO scaffold, with characteristics such as photothermal therapy to kill tumors, macrophage polarization to promote blood vessel formation, and induction of bone formation. CePO4/CS/GO scaffold activates the caspase-3 proteasein local tumor cells, thereby lysing the DNA between nucleosomes and causing apoptosis. On the one hand, the as-released Ce3+ ions promote M2 polarization of macrophages, which secretes vascular endothelial growth factor (VEGF) and Arginase-1 (Arg-1), which promotes angiogenesis. On the other hand, the as-released Ce3+ ions also activated the BMP-2/Smad signaling pathway which facilitated bone tissue regeneration. CONCLUSION: The multifunctional CePO4/CS/GO scaffolds may become a promising platform for therapy of breast cancer bone metastases.

Quercetin inhibits macrophage polarization through the p-38α/ß signalling pathway and regulates OPG/RANKL balance in a mouse skull model.

Aseptic loosening caused by wear particles is a common complication after total hip arthroplasty. We investigated the effect of the quercetin on wear particle-mediated macrophage polarization, inflammatory response and osteolysis. In vitro, we verified that Ti particles promoted the differentiation of RAW264.7 cells into M1 macrophages through p-38α/ß signalling pathway by using flow cytometry, immunofluorescence assay and small interfering p-38α/ß RNA. We used enzyme-linked immunosorbent assays to confirm that the protein expression of M1 macrophages increased in the presence of Ti particles and that these pro-inflammatory factors further regulated the imbalance of OPG/RANKL and promoted the differentiation of osteoclasts. However, this could be suppressed, and the protein expression of M2 macrophages was increased by the presence of the quercetin. In vivo, we revealed similar results in the mouse skull by µ-CT, H&E staining, immunohistochemistry and immunofluorescence assay. We obtained samples from patients with osteolytic tissue. Immunofluorescence analysis indicated that most of the macrophages surrounding the wear particles were M1 macrophages and that pro-inflammatory factors were released. Titanium particle-mediated M1 macrophage polarization, which caused the release of pro-inflammatory factors through the p-38α/ß signalling pathway, regulated OPG/RANKL balance. Macrophage polarization is expected to become a new clinical drug therapeutic target.

Ursolic acid loaded-mesoporous bioglass/chitosan porous scaffolds as drug delivery system for bone regeneration.

Bioglass scaffolds have great application potentials in orthopedics, and Ursolic acid (UA) can effectively promote in vivo new bone formation. Herein, we for the first time developed the mesoporous bioglass/chitosan porous scaffolds loaded with UA (MBG/CS/UA) for enhanced bone regeneration. The MBG microspheres with particle sizes of ~300 nm and pore sizes of ~3.9 nm were uniformly dispersed on the CS films. The mesoporous structure within the MBG microspheres and the hydrogen bonding between the scaffolds and UA drugs made the MBG/CS/UA scaffolds have controlled drug release performances. The as-released UA drugs from the scaffolds increased remarkably the alkaline phosphatase activity, osteogenic differentiation related gene type I collagen, runt-related transcription factor 2 expression, and osteoblast-associated protein expression. Moreover, the results of micro-CT images, histomorphological observations demonstrated that the MBG/CS/UA scaffolds improved new bone formation ability. Therefore, the MBG/CS/UA porous scaffolds can be used as novel bone tissue engineering materials.

Calcineurin/NFAT pathway mediates wear particle-induced TNF-α release and osteoclastogenesis from mice bone marrow macrophages in vitro.

AIM: To investigate the roles of the calcineurin/nuclear factor of activated T cells (NFAT) pathway in regulation of wear particles-induced cytokine release and osteoclastogenesis from mouse bone marrow macrophages in vitro. METHODS: Osteoclasts were induced from mouse bone marrow macrophages (BMMs) in the presence of 100 ng/mL receptor activator of NF-κB ligand (RANKL). Acridine orange staining and MTT assay were used to detect the cell viability. Osteoclastogenesis was determined using TRAP staining and RT-PCR. Bone pit resorption assay was used to examine osteoclast phenotype. The expression and cellular localization of NFATc1 were examined using RT-PCR and immunofluorescent staining. The production of TNFα was analyzed with ELISA. RESULTS: Titanium (Ti) or polymethylmethacrylate (PMMA) particles (0.1 mg/mL) did not significantly change the viability of BMMs, but twice increased the differentiation of BMMs into mature osteoclasts, and markedly increased TNF-α production. The TNF-α level in the PMMA group was significantly higher than in the Ti group (96 h). The expression of NFATc1 was found in BMMs in the presence of the wear particles and RANKL. In bone pit resorption assay, the wear particles significantly increased the resorption area and total number of resorption pits in BMMs-seeded ivory slices. Addition of 11R-VIVIT peptide (a specific inhibitor of calcineurin-mediated NFAT activation, 2.0 µmol/L) did not significantly affect the viability of BMMs, but abolished almost all the wear particle-induced alterations in BMMs. Furthermore, VIVIT reduced TNF-α production much more efficiently in the PMMA group than in the Ti group (96 h). CONCLUSION: Calcineurin/NFAT pathway mediates wear particles-induced TNF-α release and osteoclastogenesis from BMMs. Blockade of this signaling pathway with VIVIT may provide a promising therapeutic modality for the treatment of periprosthetic osteolysis.

Strontium ranelate reduces cartilage degeneration and subchondral bone remodeling in rat osteoarthritis model.

AIM: To investigate whether strontium ranelate (SR), a new antiosteoporotic agent, could attenuate cartilage degeneration and subchondral bone remodeling in osteoarthritis (OA). METHODS: Medial meniscal tear (MMT) operation was performed in adult SD rats to induce OA. SR (625 or 1800 mg·kg(-1)·d(-1)) was administered via gavage for 3 or 6 weeks. After the animals were sacrificed, articular cartilage degeneration was evaluated using toluidine blue O staining, SOX9 immunohistochemistry and TUNEL assay. The changes in microarchitecture indices and tissue mineral density (TMD), chemical composition (mineral-to-collagen ratio), and intrinsic mechanical properties of the subchondral bones were measured using micro-CT scanning, confocal Raman microspectroscopy and nanoindentation testing, respectively. RESULTS: The high-dose SR significantly attenuated cartilage matrix and chondrocyte loss at 6 weeks, and decreased chondrocyte apoptosis, improved the expression of SOX9, a critical transcription factor responsible for the expression of anabolic genes type II collagen and aggrecan, at both 3 and 6 weeks. Meanwhile, the high-dose SR also significantly attenuated the subchondral bone remodeling at both 3 and 6 weeks, as shown by the improved microarchitecture indices, TMD, mineral-to-collagen ratio and intrinsic mechanical properties. In contrast, the low-dose SR did not significantly change all the detection indices of cartilage and bone at both 3 and 6 weeks. CONCLUSION: The high-dose SR treatment can reduce articular cartilage degeneration and subchondral bone remodeling in the rat MMT model of OA.

Efficacy of zoledronic acid in treatment of teoarthritis is dependent on the disease progression stage in rat medial meniscal tear model.

AIM: To investigate whether the stage of osteoarthritis (OA) progression influenced the efficacy of the third-generation bisphosphonate zoledronic acid in a rat medial meniscal tear model. METHODS: Medial meniscal tear (MMT) was surgically induced in adult male Sprague Dawley rats. Zoledronic acid (ZOL, 100 µg/kg, sc, twice a week) was administered starting immediately, early (from 4 weeks) or late (from 8 weeks) after OA induction. The degeneration of articular cartilage was evaluated with toluidine blue O staining. Subchondral bone remodeling was evaluated with X-ray micro-CT scanning. Joint pain was measured with respect to weight-bearing asymmetry. Calcitonin gene-related peptide (CGRP) expression in dorsal root ganglia (DRGs) was examined using immunofluorescence analysis. The afferent neurons in DRGs innervating the joint were identified by retrograde labeling with fluorogold. RESULTS: Progressive cartilage loss was observed during 12 weeks after OA induction. Subchondral bone remodeling manifested as increased bone resorption at early stage (4 weeks), but as increased bone accretion at advanced stages (8 weeks). Immediately and early ZOL administration significantly improved subchondral microstructural parameters, attenuated cartilage degeneration, reduced weight-bearing asymmetry and CGRP expression, whereas the late ZOL administration had no significant effects. CONCLUSION: The stage of OA progression influences the efficacy of ZOL in treating joint degeneration and pain. To obtain the maximum efficacy, bisphosphonate treatment should be initiated in rat with early stages of OA pathogenesis.

Comparison of 3D Printing Rapid Prototyping Technology with Traditional Radiographs in Evaluating Acetabular Defects in Revision Hip Arthroplasty: A Prospective and Consecutive Study.

OBJECTIVE: To compare rapid prototyping technology (RP tech) in revision total hip arthroplasty (RTHA) with traditional examination methods and to see how they are different in evaluating acetabular anatomy and designing surgical procedure. METHODS: From February 2014 to March 2018, 43 RTHA patients with complex acetabulum defects were enrolled in this prospective study regardless of age or gender. Incomplete and unclear data were excluded. Three types of radiographic examination were performed on each patient before the revision surgery. Four groups of evaluations were designed: (i) X-ray; (ii) computed tomography (CT-scan); (iii) RP tech; and (iv) CT-aided RP tech. Discrepancies between preoperative radiographic analysis and intra-operative findings were separately compared by a team of surgeons. Premade surgical plans based on each evaluation method were compared with the final surgical procedure. The compliance of anatomic evaluation and surgical plan-design based on 3D RP tech and traditional radiographs were ranked manually by a of team surgeons into: (i) complete accordance; (ii) general accordance; and (iii) undetermined structure/procedure. The difference in ranks between RP tech and traditional radiographic methods were analyzed with a nonparametric Kruskal-Wallis test. P < 0.05 was considered significant. Multiple adjustments were taken for the statistical tests level according to the Bonferroni method. RESULTS: For anatomic analysis, the accordance in four groups of evaluating methods differed from each other (P < 0.05) except for the comparison of RP tech and CT-aided RP tech. RP tech displayed better anatomic evaluating accuracy than traditional methods (X-ray and CT) with the "complete accordance" rates of these groups being 88.37%, 4.65% and 27.91%, respectively. But CT-aided RP tech did not improve accuracy significantly compared with using RP tech individually, although the value seems high in the CT-aided RP group with the "complete accordance" rate of 95.35%. For surgery design, RP tech significantly showed better applicable surgical design compared with X-ray and CT (P < 0.05), and the "complete accordance" rates were 88.37%, 6.98% and 23.26%, but no significant difference was observed between RP tech and CT-aided RP tech, and the "complete accordance" rate of CT-aided RP tech group was 97.67%. RP tech showed remarkable improvement in bone defect assessment and surgical plan design. CONCLUSION: Using RP technology improved both sensibility and accuracy in acetabular defect evaluation with better locating and evaluating efficiency compared with X-ray and CT-scans. It also improved surgical schedule designing in complex acetabular defecting revision surgery. In particularly complex cases, CT aided RP tech may increase the accuracy of RP tech.

Impaired Autophagy in the Fibroblasts by Titanium Particles Increased the Release of CX3CL1 and Promoted the Chemotactic Migration of Monocytes.

Aseptic loosening (AL) is the most frequent cause of failure of total hip arthroplasties (THA). Prosthetic wear particle-induced monocyte recruitment to the periprosthetic tissue and subsequent inflammatory response are thought to be the major contribution to AL. Fibroblast is a dominant cell type in interfacial membrane (IFM) which is the main pathological feature of periprosthetic osteolysis in failed THAs. Considering the role of fibroblasts, as sentinel cells, in the synthesis of chemokines and regulation of inflammation, we hypothesize that fibroblasts might be involved in the monocyte recruitment in the pathogenesis of periprosthetic osteolysis associated with particle debris. This study explored the induction of fibroblasts on the monocyte recruitment. The results showed that titanium (Ti) particle-stimulated fibroblasts isolated from IFMs of loosened THAs significantly promoted the chemotactic migration of THP-1 cells by increasing the release of CX3CL1 (C-X3-C motif chemokine ligand 1). Further investigation demonstrated that Ti particle stimulation increased the expression of ADAM10 (ADAM metallopeptidase domain 10) by impairing autophagy in the fibroblasts and in turn increased the cleavage and shedding of CX3CL1. Thus, we propose a new insight to the pathogenesis of aseptic loosening which implies that autophagy-ADAM10-CX3CL1 signaling pathway in fibroblasts can be leveraged to alleviating inflammation caused by monocyte recruitment in aseptic loosening and improving performance of articulation of the joint device.

Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Improves Spinal Cord Function After Injury in Rats by Activating Autophagy.

BACKGROUND: Spinal cord injury (SCI) is a global medical problem. The smallest membrane-bound nanovesicles, known as exosomes, have a role in complex intercellular communication systems and can be used directly as therapeutic agents by acting as important paracrine factors. Nevertheless, the use of exosomes derived from BMSCs (BMSC-Exos) to treat SCI has been less, and the specific mechanism has not yet been reported. METHODS: BMSC-Exos were characterized by TEM, NTA and Western blot. The effects of BMSC-Exos treatment were compared by SCI in vivo model and a series of in vitro experiments. RESULTS: BMSC-Exos were found to enhance the expression of autophagy-related proteins LC3IIB and Beclin-1 and enabled autophagosomes formation. After BMSC-Exos treatment, there was marked decline in the level of expression of proapoptotic protein cleaved caspase-3, while that of the antiapoptotic protein Bcl-2 was upregulated. CONCLUSION: BMSC-Exos can attenuate neuronal apoptosis by promoting autophagy and promote the potential efficacy of functional behavior recovery in SCI rats. In summary, these findings expand the theoretical knowledge and forms a realistic route for the future treatment of SCI by BMSC-Exos.

The Recombinant Protein EphB4-Fc Changes the Ti Particle-Mediated Imbalance of OPG/RANKL via EphrinB2/EphB4 Signaling Pathway and Inhibits the Release of Proinflammatory Factors In Vivo.

Aseptic loosening caused by wear particles is one of the common complications after total hip arthroplasty. We investigated the effect of the recombinant protein ephB4-Fc (erythropoietin-producing human hepatocellular receptor 4) on wear particle-mediated inflammatory response. In vitro, ephrinB2 expression was analyzed using siRNA-NFATc1 (nuclear factor of activated T-cells 1) and siRNA-c-Fos. Additionally, we used Tartrate-resistant acid phosphatase (TRAP) staining, bone pit resorption, Enzyme-linked immunosorbent assay (ELISA), as well as ephrinB2 overexpression and knockdown experiments to verify the effect of ephB4-Fc on osteoclast differentiation and function. In vivo, a mouse skull model was constructed to test whether the ephB4-Fc inhibits osteolysis and inhibits inflammation by micro-CT, H&E staining, immunohistochemistry, and immunofluorescence. The gene expression of ephrinB2 was regulated by c-Fos/NFATc1. Titanium wear particles activated this signaling pathway to the promoted expression of the ephrinB2 gene. However, ephrinB2 protein can be activated by osteoblast membrane receptor ephB4 to inhibit osteoclast differentiation. In in vivo experiments, we found that ephB4 could regulate Ti particle-mediated imbalance of OPG/RANKL, and the most important finding was that ephB4 relieved the release of proinflammatory factors. The ephB4-Fc inhibits wear particle-mediated osteolysis and inflammatory response through the ephrinB2/EphB4 bidirectional signaling pathway, and ephrinB2 ligand is expected to become a new clinical drug therapeutic target.

Changes in Alignment of Ipsilateral Knee on Computed Tomography after Total Hip Arthroplasty for Developmental Dysplasia of the Hip.

OBJECTIVE: To assess the changes in alignment of ipsilateral knee joint after total hip arthroplasty (THA) for patients with developmental dysplasia of the hip (DDH). METHODS: Thirty-four patients with DDH (38 hips) who underwent THA between February and December 2008 were included in the study: 4 men and 30 women with a mean age of 56.2 years. According to Crowe classification, 11 patients were grade I, 12 were grade II, 9 were grade III, and 6 were grade IV. Computed tomography scans were performed from the anterior superior iliac spine to the tibial tubercle before surgery and at last follow-up. Femoral anteversion angle, leg lengthening, and knee alignment, including patellar tilt angle, lateral patellar displacement, and tibiofemoral rotation angle, were measured on computed tomography scans, and their relationships were analyzed. RESULTS: The mean follow-up period was 51.5 months (range, 39-70 months). There were no intraoperative fractures, and no infections occurred during the follow-up period. One patient developed deep venous thrombosis and another suffered from femoral nerve palsy. The mean preoperative Harris Hip Score was 48.9 ± 7.5 and improved to 91.2 ± 8.3 by the last follow-up (P < 0.001). There was no sign of prosthetic loosening in all hips. Postoperatively, mean leg lengthening was 26.08 ± 21.81 mm (P < 0.001), femoral anteversion decreased 9.03° ± 12.80° (P < 0.001), and patellar tilt, lateral patellar displacement, and tibiofemoral rotation increased by 3.58° ± 4.96° (P < 0.001), 1.78 ± 3.36 mm (P = 0.002), and 2.56° ± 3.37° (P < 0.001), respectively. Postoperative increase in patellar tilt and lateral patellar displacement had significant linear relationships with the decrease in femoral anteversion (r = 0.621, P < 0.001 and r = 0.437, P = 0.0037, respectively). These results revealed that patellofemoral alignment would change more with the decrease in femoral anteversion. Postoperative increase in external rotation of the tibia had significant positive linear relationships with leg lengthening (r = 0.34, P = 0.037) and the decrease in femoral anteversion (r = 0.693, P < 0.001). These results revealed that the external rotation of the proximal tibia would increase with the leg lengthening or the decrease of femoral anteversion. Postoperative changes in patellar tilt and lateral patellar displacement had no significant linear relationships with leg lengthening (P = 0.795 and P = 0.082, respectively). CONCLUSIONS: Total hip arthroplasty for DDH could induce changes in alignment of ipsilateral patellofemoral and tibiofemoral joints, with increases in patellar tilt and displacement, and increases in external rotation of the tibia. These secondary alterations still existed at medium-term follow-up after surgery, which should be considered during THA for patients with DDH. Extended follow-up is necessary to evaluate long-term changes in the knee joint.

Titanium particle­mediated osteoclastogenesis may be attenuated via bidirectional ephrin­B2/eph­B4 signaling in vitro.

The present study investigated the role of bidirectional ephrin­B2/erythropoietin­producing human hepatocellular receptor 4 (ephB4) signaling in the regulation of wear particle­mediated osteoclastogenesis in vitro. Mouse bone marrow macrophages (BMMs) were induced into osteoclasts by receptor activator of nuclear factor­κB ligand (RANKL, 50 ng/ml). EphB4­Fc, an osteoblast membrane surface receptor (4 µg/ml), was used to stimulate the ephrin­B2 ligand of osteoclasts in the presence and absence of titanium (Ti). Tartrate­resistant acid phosphatase (TRAP) staining was used to detect the number of osteoclasts, and phalloidin staining was used to examine the cytoskeletons of the osteoclasts. A bone pit absorption experiment was used to measure osteoclast function. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to examine osteoclastogenesis. ELISAs were used to detect the production of inflammatory factors. The data demonstrated that Ti significantly promoted the differentiation of BMMs into mature osteoclasts in the presence of RANKL and significantly promoted expression of the ephrin­B2, nuclear factor of activated T­cells 1 (NFATc1), TRAP, Fos proto­oncogene, AP­1 transcription factor subunit (C­FOS), and matrix metalloproteinase 9 (MMP9) genes. Phalloidin and TRAP staining revealed that following the addition of ephB4­Fc, the number, size and cytoskeletal elements of osteoclasts were significantly decreased compared with those in the titanium particle group without ephB4­Fc. Compared with the titanium particle group, the bone pit absorption experiment revealed significantly decreased absorption pit areas in the titanium particle+ephB4­Fc group. The expression of the NFATc1, TRAP, C­FOS and MMP9 genes was markedly decreased in the ephB4­Fc group; however, the expression of the ephrin­B2 gene was increased compared with the Ti particle group without ephB4­Fc after 5 days. Production of inflammatory cytokines was inhibited by Ti particles through bidirectional signals. Addition of ephB4­Fc inhibited the osteoclast­mediated formation of Ti particles via bidirectional ephrin­B2/ephB4 signaling. Activation of this bidirectional signaling pathway may be a potential clinical treatment for osteolysis surrounding prostheses.

In vitro study of knee stability after two-band two-tunnel posterior cruciate ligament reconstruction.

OBJECTIVE: To compare the ability of three different reconstruction procedures in restoring the posterior displacement of tibia and the posterior stability of the knee joint from 0 degree to 120 degrees flexion. METHODS: Three posterior cruciate ligaments (PCL) reconstruction procedures were performed, namely two-band two-tunnel reconstruction, one-band anterior tunnel reconstruction and one-band posterior tunnel reconstruction. The posterior displacement of the tibia in relation to the femur was measured when a 200N posterior force was applied. RESULTS: Within the flexion range of 0 degree to 30 degrees, the displacement in the one-band posterior tunnel reconstruction showed little difference from that of an intact knee (P>0.05). But when the flexion exceeded 30 degrees, especially when it exceeded 60 degrees, the displacement in one-band posterior tunnel reconstruction was much greater than that of an intact knee (P<0.01). In two-band two-tunnel reconstruction and one-band anterior tunnel reconstruction, the displacement was approximately the same as that of an intact knee ranging from 0 degree to 120 degrees (P>0.05), while a slight over-restriction might be found at some angles. CONCLUSIONS: Two-band reconstruction could effectively restrict the posterior displacement of the tibia and restore anterior, posterior stability of the knee joint within its full range of flexion. One-band anterior tunnel reconstruction also could maintain the posterior stability of the knee, while the result of one-band posterior tunnel reconstruction is the most unsatisfactory.

[The effect of ultra high molecular weight polyethylene particles on macrophage].

OBJECTIVE: To study the effect of ultra high molecular weight polyethylene (UHMWPE) particles on macrophages and evaluate the expression of NFAT2, a key transcriptional factor for osteoclast differentiation. METHODS: From November 2004 to February 2005, macrophages were co-cultured with UHMWPE particles. When observed at different times, the proliferation activity of macrophages was analyzed by MTT and the expression of calcineurin (CaN) and NFAT2 by immunohistochemical and RT-PCR method respectively. RESULTS: The macrophages phagocytosed UHMWPE particles in an early time, the expression of CaN and NFAT2 was increased, while the proliferation activity was not enhanced. CONCLUSIONS: UHMWPE particles can stimulate macrophages to phagocytose significantly, and enhance the expression of the transcriptional factor NFAT2.

Dynamic Alterations in Microarchitecture, Mineralization and Mechanical Property of Subchondral Bone in Rat Medial Meniscal Tear Model of Osteoarthritis.

BACKGROUND: The properties of subchondral bone influence the integrity of articular cartilage in the pathogenesis of osteoarthritis (OA). However, the characteristics of subchondral bone alterations remain unresolved. The present study aimed to observe the dynamic alterations in the microarchitecture, mineralization, and mechanical properties of subchondral bone during the progression of OA. METHODS: A medial meniscal tear (MMT) operation was performed in 128 adult Sprague Dawley rats to induce OA. At 2, 4, 8, and 12 weeks following the MMT operation, cartilage degeneration was evaluated using toluidine blue O staining, whereas changes in the microarchitecture indices and tissue mineral density (TMD), mineral-to-collagen ratio, and intrinsic mechanical properties of subchondral bone plates (BPs) and trabecular bones (Tbs) were measured using micro-computed tomography scanning, confocal Raman microspectroscopy and nanoindentation testing, respectively. RESULTS: Cartilage degeneration occurred and worsened progressively from 2 to 12 weeks after OA induction. Microarchitecture analysis revealed that the subchondral bone shifted from bone resorption early (reduced trabecular BV/TV, trabecular number, connectivity density and trabecular thickness [Tb.Th], and increased trabecular spacing (Tb.Sp) at 2 and 4 weeks) to bone accretion late (increased BV/TV, Tb.Th and thickness of subchondral bone plate, and reduced Tb.Sp at 8 and 12 weeks). The TMD of both the BP and Tb displayed no significant changes at 2 and 4 weeks but decreased at 8 and 12 weeks. The mineral-to-collagen ratio showed a significant decrease from 4 weeks for the Tb and from 8 weeks for the BP after OA induction. Both the elastic modulus and hardness of the Tb showed a significant decrease from 4 weeks after OA induction. The BP showed a significant decrease in its elastic modulus from 8 weeks and its hardness from 4 weeks. CONCLUSION: The microarchitecture, mineralization and mechanical properties of subchondral bone changed in a time-dependent manner as OA progressed.

Effect of impaction on gene-modified cells seeded on granular bone allografts in vitro and in vivo.

BACKGROUND: While attempting to restore bone stock, impaction bone grafting employed during revision joint surgery may result in slow and limited allograft incorporation into host bone. A new approach including gene-modified bone marrow stromal cells (BMSCs) in combination with impaction bone grafting may effectively restore bone stock and improve allograft incorporation. This study aimed to investigate the effect of impaction on gene-modified BMSCs seeded on granular bone allografts in vitro and in vivo. METHODS: Deep-frozen, granular, cancellous bone allografts from canines were prepared to serve as cell delivery scaffolds and were seeded with green fluorescent protein (GFP) genetically-modified BMSCs to construct cell-allograft composites. The composites were impacted in a simulative, in vitro impaction model and cultured for further analysis under standard conditions. Four Beagle dogs, treated with bilateral, uncemented proximal tibial joint hemiarthroplasty with a prosthesis, were implanted with autologous GFP gene-modified cell-allograft composites to repair the bone cavity around each prosthesis. RESULTS: A significant reduction in cell viability was observed after impaction by fluorescence microscopy in vitro. However, there remained a proportion of GFP-positive cells that were viable and functionally active, as evidenced by the secretion of GFP protein in vitro and in vivo. CONCLUSIONS: Gene-modified BMSCs seeded on granular allografts were able to withstand the impaction forces and to maintain their normal functions in vitro and in vivo, in spite of a partial loss in cell viability.