Zhinong Huang

Modulating osteogenesis of mesenchymal stem cells by modifying growth factor availability

Growth factors control the proliferation and differentiation of osteoprogenitor cells. This study explores the effects of modulating growth factors (VEGF, IGF-1, FGF-2 and BMP-2) on osteogenesis of mesenchymal stem cells (MSCs) in vitro. Constant and profiled delivery protocols, in accordance with protein expression in vitro, were applied to deliver or neutralize growth factors. Cell number, alkaline phosphatase (ALP-2) and osteocalcin (OC) expression, and mineralization were measured as outcome variables. Profiled addition of VEGF increased MSC proliferation. Constant and profiled application of FGF-2 and neutralization of IGF-1 and BMP-2 decreased ALP-2 levels. Profiled addition of BMP-2 vastly increased OC release from MSCs, but constant addition of IGF-1, constant and profiled neutralization of IGF-1 and FGF-2 reduced OC levels. Constant addition of IGF-1 and FGF-2, as well as profiled loading of FGF-2 decreased mineralization of MSCs. This study indicated that endogenous IGF-1 and FGF-2 are essential to osteogenesis; excess IGF-1 and FGF-2 were inhibitory to bone formation. Selective, temporally specific addition of growth factors, such as BMP-2 and VEGF appears to be an important strategy to enhance osteogenesis.

Effects of orthopedic polymer particles on chemotaxis of macrophages and mesenchymal stem cells

Wear particles generated from total joint arthroplasty (TJA) stimulate macrophages to release chemokines. The role of chemokines released from wear particle-stimulated macrophages on the migration of macrophages and osteoprogenitor cells in vitro has not been elucidated. In this study, we challenged murine macrophages (RAW 264.7) with clinically relevant polymethyl methacrylate (PMMA, 1-10 microm) and ultra high molecular weight polyethylene (UHMWPE, 2-3 microm) particles. The chemotactic effects of the conditioned media (CM) were tested in vitro using human macrophages (THP-1) and human mesenchymal stem cells (MSCs) as the migrating cells. CM collected from both particle types had a chemotactic effect on human macrophages, which could be eliminated by monocyte chemotactic protein-1 (MCP-1) neutralizing antibody. Blocking the CCR1 receptor eliminated the chemotactic effect, while CCR2 antibody only partially decreased THP-1 cell migration. CM from PMMA but not UHMWPE-exposed macrophages led to chemotaxis of MSCs; this effect could be eliminated by macrophage inflammatory protein-1 alpha (MIP-1alpha) neutralizing antibody. Neither CCR1 nor CCR2 blocking antibodies showed an effect on the migration of MSCs. Chemokines released by macrophages stimulated by wear particles can have an effect on the migration of macrophages and MSCs. This effect seems to be dependent on the particle type, and may be modulated by MCP-1 and MIP-1alpha, however, more than one chemokine may be necessary for chemotaxis.

An in vivo murine model of continuous intramedullary infusion of polyethylene particles

Wear debris affects both initial osseointegration and subsequent bone remodeling of total joint replacements (TJRs). To study the complex cascade associated with the continuous generation of particles, a robust animal model is essential. To date, an animal model that incorporates continuously delivered particles to an intramedullary orthopaedic implant has not been available. In this study, we successfully infused clinically relevant ultra high molecular weight polyethylene particles, previously isolated from joint simulator tests, to the intramedullary space of the mouse femur for 4 weeks using a subcutaneous osmotic pump. Reduction of bone volume following the 4-week infusion of UHMWPE was detected by microCT. UHMWPE particles also changed the level of Alkaline Phosphatase expression in the infused femurs. Continuous infusion of particles to the murine bone-implant interface simulated the clinical scenario of local polymer wear particle generation and delivery in humans and can be used to further study the biological processes associated with wear debris particles.

Effect of Nanofiber-Coated Surfaces on the Proliferation and Differentiation of Osteoprogenitors In Vitro

The osteoconductive property of titanium (Ti) surfaces is important in orthopedic and dental implant devices. Surface modifications of Ti have been proposed to further improve osseointegration. In this study, three different materials, silicon (Si), silicon oxide (SiO(2)), and titanium oxide (TiO(2)), were used to construct nanofibers for surface coating of Ti alloy Ti-6Al-4 V (Ti alloy). MC3T3-E1 osteoprogenitor cells were seeded on nanofiber-coated discs and cultured for 42 days. DNA, alkaline phosphatase, osteocalcin, and mineralization nodules were measured using PicoGreen, enzyme-linked immunosorbent assay, and calcein blue staining to detect the attachment, proliferation, differentiation, and mineralization of MC3T3-E1 cells, respectively. The results demonstrated that the initial cell attachments on nanofiber-coated discs were significantly lower, although cell proliferation on Si and SiO(2) nanofiber-coated discs was better than on Ti alloy surfaces. TiO(2) nanofibers facilitated a higher cellular differentiation capacity than Ti alloy and tissue culture-treated polystyrene surfaces. Thus, surface modification using nanofibers of various materials can alter the attachment, proliferation, and differentiation of osteoprogenitor cells in vitro.

Surveillance of systemic trafficking of macrophages induced by UHMWPE particles in nude mice by noninvasive imaging

Macrophages constitute a major part of the cell response to wear particles produced at articulating and nonarticulating interfaces of joint replacements. This foreign body reaction can result in periprosthetic osteolysis and implant loosening. We demonstrate that ultra-high molecular weight polyethylene (UHMWPE) particles induce systemic trafficking of macrophages by noninvasive in vivo imaging and immunohistochemistry. The distal femora of nude mice were injected with 60 mg/mL UHMWPE suspension or saline alone. Reporter RAW264.7 macrophages that stably expressed the bioluminescent reporter gene and the fluorescence reporter gene were injected intravenously. Bioluminescence imaging was performed using an in vivo imaging system immediately after macrophage injection and at 2-day intervals. Compared with the nonoperated contralateral femora, at day 4, 6, and 8, the bioluminescent signal of femora containing UHMWPE suspension increased 1.30 +/- 0.09-, 2.36 +/- 0.92-, and 10.32 +/- 7.61-fold, respectively. The values at same time points for saline-injected control group were 1.08 +/- 0.07-, 1.14 +/- 0.27-, and 1.14 +/- 0.35-fold, respectively. The relative bioluminescence of the UHMWPE group was higher at all postinjection days and significantly greater than the saline group at day 8 (p < 0.05). Histological analysis confirmed the presence of reporter macrophages within the medullary canal of mice with implanted UHMWPE particles. The presence of UHMWPE particles induced enhanced bone remodeling activity. Clinically relevant UHMWPE particles stimulated the systemic recruitment of macrophages during an early time course using the murine femoral implant model. Interference with systemic macrophage trafficking may potentially mitigate UHMWPE particle-induced periprosthetic osteolysis.

In vivo murine model of continuous intramedullary infusion of particles—A preliminary study

Continued production of wear debris affects both initial osseointegration and subsequent bone remodeling of total joint replacements (TJRs). However, continuous delivery of clinically relevant particles using a viable, cost effective, quantitative animal model to simulate the scenario in humans has been a challenge for orthopedic researchers. In this study, we successfully infused blue-dyed polystyrene particles, similar in size to wear debris in humans, to the intramedullary space of the mouse femur for 4 weeks using an osmotic pump. Approximately 40% of the original particle load (85 microL) was delivered into the intramedullary space, an estimate of 3 x 10(9) particles. The visible blue dye carried by the particles confirmed the delivery. This model demonstrated that continuous infusion of particles to the murine bone-implant interface is possible. In vivo biological processes associated using wear debris particles can be studied using this new animal model.

Polymethylmethacrylate particle exposure causes changes in p38 MAPK and TGF-β signaling in differentiating MC3T3-E1 cells

Periprosthetic osteolysis of joint replacements caused by wear debris is a significant complication of joint replacements. Polymethylmethacrylate (PMMA) particles have been shown to inhibit osteogenic differentiation, but the molecular mechanism has not been previously determined. In this study, we exposed differentiating MC3T3-E1 preostoblast cells to PMMA particles and determined the changes that occurred with respect to p38 mitogen-activated protein kinase (MAPK) activity and the transforming growth factor (TGF)-beta1 and bone morphogenetic protein (BMP) signaling pathways. In the absence of particles, MC3T3-E1 cells demonstrate activation of p38 MAPK on day 8 of differentiation; however, when treated with PMMA particles, differentiating MC3T3-E1 cells demonstrate the suppression of p38 activity on day 8 and show activation of p38 on days 1 and 4. On day 4 of particle exposure, the differentiating MC3T3-E1 cells show significant downregulation of TGF-beta1 expression, which is involved in osteoblast differentiation, and a significant upregulation of the expression of BMP3 and Sclerostin (SOST), which are negative regulators of osteoblast differentiation. By day 8 of particle exposure, the changes in TGF-beta1, BMP3, and SOST expression are opposite of those seen on day 4. This study has demonstrated the distinct changes in the molecular profile of MC3T3-E1 cells during particle-induced inhibition of osteoblast differentiation. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

New bone formation by murine osteoprogenitor cells cultured on corticocancellous allograft bone

The gold standard for bone grafting in orthopedics is autograft, however autograft has a limited supply and is associated with significant morbidity at the harvest site. One alternative, allograft bone, provides an osteoconductive scaffold, is in less limited supply, and it does not require a harvest from the patient. However, allograft lacks both osteogenic cells and osteoinductive proteins that make autograft bone so advantageous. This study provides a model to investigate strategies for augmentation of corticocancellous allograft bone discs with bone marrow‐derived osteoprogenitor cells (OPCs) plus exogenous growth factors in vitro. In this model, allograft bone discs were created by cutting 1‐mm thick slices from the distal femur and proximal tibia of euthanized mice. The allografts were sterilized and scanned by micro‐computed tomography (µCT) to provide the pre‐culture graft volume and trabecular characteristics. The discs were then seeded with OPCs harvested from murine bone marrow. The seeded grafts were placed in organ culture until harvest, after which they were re‐scanned by µCT and the data compared to the corresponding pre‐culture data. In addition, bone morphogenetic protein‐7 (BMP‐7, also know as osteogenic protein‐1 or OP‐1), basic fibroblast growth factor (bFGF), and OP‐1 combined with bFGF were added on a daily basis to the cultures. After final µCT scanning, all grafts were sectioned and evaluated histologically after hematoxylin and eosin (H&E) staining. µCT scans of cultured allografts with cells at 3, 5, and 9 weeks showed a time‐dependent, statistically significant increase in bone volume. The trabecular thickness (Tb.Th.) of grafts, from both groups that were augmented with OP‐1, showed a statistically significant increase in trabecular thickness of allografts with OPCs. These data suggest that bone marrow‐derived OPCs adhere to, and produce, new bone on corticocancellous allograft in vitro. When exogenous OP‐1 is added to this model, an increase in the production of bone onto the corticocancellous allograft bone disc is seen. This model allows for the investigation of the effects of multiple growth factors, and other interventions, on OPCs seeded onto allograft bone in vitro.

Molecular profile of osteoprogenitor cells seeded on allograft bone.

In order to optimize and modulate bone formation it is essential to understand the expression patterns of key bone‐specific growth factors, as osteoprogenitor cells undergo the processes of proliferation, differentiation and maturation. This study reports the sequential expression of bone‐related growth and transcription factors when bone marrow‐derived osteoprogenitor cells from C57BL mice were cultured on allograft bone discs. Mineralization and osteocalcin protein levels were used to track osteogenic differentiation and maturation. Bone‐related growth factors, such as Bmp‐2, Bmp‐7, Ctnnb‐1, Fgf‐2, Igf‐1, Vegf‐a and Tgf‐β1, and transcription factors, such as Runx‐2 and osteocalcin, were examined by enzyme‐linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT‐PCR). Total density of mineralized bone was significantly increased 7.6 ± 0.7% in allografts cultured with cells, compared with a 0.5 ± 2.0% increase in the controls without cells (p < 0.01). Osteocalcin protein levels peaked at day 4. Protein expression showed peaks of BMP‐2 and TGF‐β1 on day 2, with VEGF peaking on day 8, and IGF‐1 decreasing on day 2. mRNA for Pdgf‐a peaked on day 2; Bmp‐2 on days 4 and 16; Ctnnb‐1 on days 8 and 20; Vegf‐a, Fgf‐2, Runx‐2 and Igf‐1 on day 12; Tgf‐β1 on day 16; and Pdgf‐b on day 20. Osteogenic growth factors correlated with Runx‐2 and Ctnnb‐1, whereas a predominant vascular growth factor, Vegf‐a, did not follow this pattern. Specific bone‐related genes and proteins were expressed in a time‐dependent manner when osteoprogenitor cells were cultured on cortico‐cancellous bone discs in vitro. Copyright

Quantitation of Bone Area in Undecalcified Frozen Sections With Fluorescent Microscopy

Abstract Histomorphometry is a useful tool for assessing the dynamics of bone in orthopaedic research. We compared a new fluorescence imaging method with a commonly used computer-interactive manual method using hematoxylin and eosin staining to quantify the total bone area in 6-μm thick frozen undecalcified sections processed from a bone chamber. Eighteen sections were analyzed by both methods, and the correlation coefficient was 0.92. The advantages of the fluorescence imaging are that it is simple, objective, and correlates strongly with traditional more tedious methods for histomorphometric analysis of bone. (The J Histotechnol 31:15, 2008) Submitted July 24, 2007; accepted with revisions January 9, 2008