Device-associated bacterial infections are a major and costly clinical challenge. This project aimed to develop a smart new biomaterial for implants that helps to protect against infection and inflammation, promote bone growth, and is biodegradable. Gallium (Ga) doped strontium-phosphate was coated on pure Magnesium (Mg) through a chemical conversion process. Mg was distributed in a graduated manner throughout the strontium-phosphate coating GaSrPO4, with a compact structure and a Ga-rich surface. We tested this sample for its biocompatibility, effects on bone remodeling and antibacterial activities including Staphylococcus aureus, S. epidermidis and E. coli - key strains causing infection and early failure of the surgical implantations in orthopaedics and trauma.

Ga was distributed in a gradient way throughout the entire strontium-phosphate coating with a compact structure and a gallium-rich surface. The GaSrPO4 coating protected the underlying Mg from substantial degradation in minimal essential media at physiological conditions over 9 days. The liberated Ga ions from the coatings upon Mg specimens inhibited the growth of bacterial tested. The Ga dopants showed minimal interferences with the SrPO4 based coating, which boosted osteoblasts and undermined osteoclasts in in vitro co-cultures model.

The results evidenced this new material may be further translated to preclinical trial in large animal model and towards clinical trial.

Acknowledgements: Authors are grateful to the financial support from the Australian Research Council through the Linkage Scheme (ARC LP150100343). The authors acknowledge the facilities, and the scientific and technical assistance of the RMIT University and John Curtin School of Medical Research, Australian National University.

Infection of implanted medical devices (biomaterials), like titanium orthopaedic implants, can have disastrous consequences, including removal of the device. These so-called biomaterial-associated infections (BAI) are mainly caused by Staphylococcus aureus and Staphylococcus epidermidis. To prevent biofilm formation using a non-antibiotic based strategy, we aimed to develop a novel permanently fixed antimicrobial coating for titanium devices based on stable immobilized quaternary ammonium compounds (QACs).

Medical grade titanium implants were dip-coated in subsequent solutions of hyperbranched polymer, polyethyleneimine and 10 mM sodium iodide, and ethanol. The QAC-coating was characterized using water contact angle measurements, scanning electron microscopy, FTIR, AFM and XPS. The antimicrobial activity of the coating was evaluated against S. aureus strain JAR060131 and S. epidermidis strain ATCC 12228 using the JIS Z 2801:2000 surface microbicidal assay. Lastly, we assessed the in vivo antimicrobial activity in a mouse subcutaneous implant infection model with S. aureus administered locally on the QAC-coated implants prior to implantation to mimic contamination during surgery.

Detailed material characterization of the titanium samples showed the presence of a homogenous and stable coating layer at the titanium surface. Moreover, the coating successfully killed S. aureus and S. epidermidis in vitro. The QAC-coating strongly reduced S. aureus colonization of the implant surface as well as of the surrounding tissue, with no apparent macroscopic signs of toxicity or inflammation in the peri-implant tissue at 1 and 4 days after implantation.

An antimicrobial coating with stable quaternary ammonium compounds on titanium has been developed which holds promise to prevent BAI. Non-antibiotic-based antimicrobial coatings have great significance in guiding the design of novel antimicrobial coatings in the present, post-antibiotic era.

Acknowledgements: This research was financially supported by the Health∼Holland/LSH-TKI call 2021–2022, project 25687, NACQAC: ‘Novel antimicrobial coatings with stable non-antibiotic Quaternary Ammonium Compounds and photosensitizer technology'.

Aim

The use of medical devices has grown significantly over the last decades, and has become a major part of modern medicine and our daily life. Infection of implanted medical devices (biomaterials), like titanium orthopaedic implants, can have disastrous consequences, including removal of the device. For still not well understood reasons, the presence of a foreign body strongly increases susceptibility to infection. These so-called biomaterial-associated infections (BAI) are mainly caused by Staphylococcus aureus and Staphylococcus epidermidis. Formation of biofilms on the biomaterial surface is generally considered the main reason for these persistent infections, although bacteria may also enter the surrounding tissue and become internalized within host cells. To prevent biofilm formation using a non-antibiotic based strategy, we aimed to develop a novel permanently fixed antimicrobial coating for titanium devices based on stable immobilized quaternary ammonium compounds (QACs).

Hip and knee arthroplasty (HKA) are two of the most successful orthopaedic procedures. However, one major complication necessitating revision surgery is osteolysis causing aseptic loosening of the prosthesis. JAK-STAT has been demonstrated to influence bone metabolism and can be regulated by microRNA (miRNA).

Adult patients with osteolysis or aseptic loosening undergoing revision HKA were recruited. Age and gender matched patients undergoing primary hip or knee arthroplasty were our controls. Samples of bone, tissue and blood were collected and RNA isolation was performed. The best quality samples were used for RNA-sequencing. Data analysis was performed using RStudio and Galaxy to identify differentially expressed genes. Western blotting of IL6 was used to confirm protein expression.

Five circulating miRNA were identified which had 10 differentially expressed genes in bone and 11 differentially expressed genes in tissue related to the JAK-STAT pathway. IL6 in bone and EpoR in bone were highly significant and IL6 in tissue, MPL in bone, SOCS3 in tissue, JAK3 in bone and SPRED1 in bone were borderline significant. Western blot results demonstrated up-expression of IL6 in bone tissue of revision patients.

Periprosthetic osteolysis and aseptic loosening can be attributed to miRNA regulation of the JAK-STAT pathway in osteoblasts and osteoclasts, leading to increased bone resorption. These findings can be used for further experiments to determine utility in the clinical setting for identifying diagnostic markers or therapeutic targets.

Aim

The purpose of this study is to report the overall infection control rate and prognostic factors associated with acute, hematogenous and chronic PJIs treated with DAIR.

Aims

The aim of this study was to further evaluate the accuracy of ten promising synovial biomarkers (bactericidal/permeability-increasing protein (BPI), lactoferrin (LTF), neutrophil gelatinase-associated lipocalin (NGAL), neutrophil elastase 2 (ELA-2), α-defensin, cathelicidin LL-37 (LL-37), human β-defensin (HBD-2), human β-defensin 3 (HBD-3), D-dimer, and procalcitonin (PCT)) for the diagnosis of periprosthetic joint infection (PJI), and to investigate whether inflammatory joint disease (IJD) activity affects their concentration in synovial fluid.

Aims

The purpose of this study was to validate our hypothesis that centrifugation may eliminate false-positive leucocyte esterase (LE) strip test results caused by autoimmune diseases in the diagnosis of knee infection.

Summary Statement

Using the latest Next Generation Sequencing technologies, we have investigated miRNA expression profiles in human trabecular bone from total hip replacement (THR) revision surgery where wear particle associated osteolysis was evident.

Purpose

Angiogenesis and osteogenesis are essential for bone growth, fracture repair, and bone remodeling. VEGF has an important role in bone repair by promoting angiogenesis and osteogenesis. In our previous study, endothelial progenitor cells (EPCs) promoted bone healing in a rat segmental bone defect as confirmed by radiological, histological and microCT evaluations (Atesok, Li, Schemitsch 2010); EPC treatment of fractures resulted in a significantly higher strength by biomechanical examination (Li, Schemitsch 2010). In addition, cell-based VEGF gene transfer has been effective in the treatment of segmental bone defects in a rabbit model (Li, Schemitsch et al 2009); Purpose of this study: Evaluation of VEGF gene expression after EPC local therapy for a rat segmental bone defect.

Purpose: The purpose of this study was to compare the effects of two types of stem/progenitor cells on the healing of critical sized bone defects in a rat model. Endothelial Progenitor Cells (EPCs), a novel cell type with previously demonstrated effects on angiogenesis in animal models of vascular disease, were compared to both a control group of no cell therapy, and a treatment group of Mesenchymal Stem Cells (MSCs). The hypothesis was that EPCs would demonstrate both superior bone healing and angiogenesis, when compared to the control group and MSC group.

Method: EPCs and MSCs were isolated from the bone marrow of syngeneic rats by differential culture and grown ex vivo for 10 days. Subsequently the cells were harvested, seeded on a gelfoam scaffold, and implanted into a 5mm segmental defect in a rat femur that had been stabilized with a plate and screws. Bone healing was assessed radiographically and by microCT. Angiogenesis was assessed by histology and physiologically, using laser doppler to assess blood flow in the bone and soft tissues. All animal protocols were approved by and performed in accordance with the St. Michael’s Hospital Animal Care Committee. ANOVA was used to test for significant differences between the groups, and a p-value of < 0.05 was considered statistically significant.

Results: The EPC (n=14) group demonstrated radiographic evidence of healing of the bone defect as early as 2 weeks, and all specimens were radiographically healed at 6 weeks. Both the control group (n=14) and the MSC group (n=14) showed no radiographic evidence of healing at 10 weeks. MicroCT comparison of the EPC group versus the control group showed significantly greater bone volume and density at the defect site (p< 0.001). More blood vessel formation was observed in the EPC group versus the control group on histology at 2 weeks. Laser Doppler assessment showed significantly more soft tissue and bone blood flow at 2 and 3 weeks in the EPC group versus the control group (p=0.021).

Conclusion: The results of this study demonstrate that EPCs are effective as cell-based therapy for healing critical sized bone defects in a rat model. In this model EPCs demonstrated superiority to MSCs with regard to bone healing. In addition, EPCs demonstrated superior angiogenesis over controls in a rat model of fracture healing. These results strongly suggest that EPCs are effective for therapeutic angiogenesis and osteogenesis in fracture healing. There is a clinical need for effective strategies in the management of traumatic bone defects and nonunions. Investigation into the use of MSCs as an effective alternative to autologous bone grafting has failed to translate into clinical use. It is possible that EPCs are more effective at the regeneration of bone in segmental defects because of their synergistic effect on angiogenesis and osteogenesis. Further research into EPC based therapies for fracture healing is warranted.

Purpose: Severe fractures damage blood vessels and disrupt circulation at the fracture site resulting in an increased risk of poor fracture healing. Endothelial progenitor cells (EPCs) are bone-marrow derived cells with the ability to differentiate into endothelial cells and contribute to neovascularization and re-endothelialization after tissue injury and ischemia. We have previously reported that EPC therapy resulted in improved radiographic healing and histological blood vessel formation in a rat fracture model. The purpose of this study was to further quantify the effects of EPC therapy with microCT and biomechanical analyses.

Method: Five-millimeter segmental defects were created and stabilized in the femora of 14 fisher 344 rats. The treatment group (n=7) received 1x106 EPCs within gelfoam locally at the area of the bone defect and control animals (n=7) received only saline-gelfoam with no cells. The formation and healing of bone after 10 weeks were asessed by radiographic, micro-CT and biomechanical analyses.

Results: Radiographically all the animals in EPC-treated group healed with bridging callus formation, whereas control group animals demonstrated radiographic non-union. Micro-CT assessment demonstrated significantly improved parameters of bone volume (35.34 to 20.68 mm³, p=0.000), bone volume density (0.24 to 0.13%, p=0.001), connectivity density (25.13 to 6.15%, p=0.030), trabecular number (1.14 to 0.51 1/mm, p=0.000), trabecular thickness (0.21 to 0.26 mm, p=0.011), trabecular spacing (0.71 to 1.88 mm, p=0.002), bone surface area (335.85 to 159.43mm, p=0.000), and bone surface to bone volume ratio (9.43 to 7.82 1/mm, p=0.013) in the defect site for the EPC group versus the control group respectively. Biomechanical testing showed that the EPC treatment group had a significantly higher torsional strength compared with the control group (EPC=164.6±27.9 Nmm, Control=29.5±3.8 Nmm; p value = 0.000). Similarly, the EPC treated fractures demonstrated significantly higher torsional stiffness versus controls (EPC=30.3±5.0 Nmm/ deg, Control=0.9±0.1 Nmm/deg; p value = 0.000). When biomechanically compared to contralateral intact limbs, the EPC treated limbs had similar torsional stiffness (p=0.996), but significantly lower torsional strength (p=0.000) and smaller angle of twist (p=0.002).

Conclusion: These results suggest that local EPC therapy significantly enhances fracture healing in an animal model. The biomechanical results show that control animals develop a mechanically unstable non-union. In contrast, EPC therapy results in fracture healing that restores the biomechanical properties of the fractured bone closer to that of intact bone.

Purpose: Vascular Endothelial Growth Factor (VEGF) plays an important role in promoting angiogenesis and osteogenesis during fracture repair. Our previous studies have shown that cell-based VEGF gene therapy accelerates bone healing of a rabbit tibia segmental bone defect in-vivo, and increases osteoblast proliferation and mineralization in-vitro. The aim of this project was to examine the effect of exogenous human VEGF (hVEGF) on the endogenous rat VEGF messenger RNA (mRNA) expression in a cell-based gene transfer model.

Method: The osteoblasts were obtained from the rat periosteum. The fibroblasts were obtained from the rat dermal tissue. The cells were then cultured to reach 60% confluence and transfected with hVEGF using Superfect. Four groups were:

osteoblast-hVEGF,

The cultured cells were harvested at 1, 3 and 7 days after the transfection. The total mRNA was extracted (TRIZOL); both hVEGF and rat VEGF mRNA were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantified by VisionWorksLS.

Results: The hVEGF mRNA was detected by RT-PCR from transfected osteoblasts after three days of gene transfection. The hVEGF mRNA expression in transfected fibroblasts increased exponentially at days 1, 3 and 7 after the transfection. We compared the endogenous rat VEGF mRNA expression level of the osteoblasts or fibroblasts that were transfected with hVEGF with the cells without the transfection. The hVEGF transfected osteoblasts had a greater rat VEGF mRNA expression than the non-transfected osteoblasts. Furthermore, when hVEGF was transfected to the rat fibroblasts, the endogenous mRNA expression level measured was also greater than that from the non-transfected fibroblasts. Rat VEGF mRNA expression increased in the first three days of the hVEGF transfection, but the expression level was reduced at Day 7.

Conclusion: These results suggest that cell-based hVEGF gene therapy enhances endogenous rat VEGF mRNA expression in both osteoblasts and fibroblasts.

Purpose: Endothelial Progenitor Cells (EPCs) have been proven to contribute to formation of new blood vessels. The objective of this study was to evaluate the effects of local EPC therapy on the stimulation of angiogenesis at a fracture site and the promotion of bone healing by increasing osteogenesis and callus formation.

Method: Rat bone marrow EPCs were isolated and cultured. A segmental bone defect (4mm.) was created in the rat femur diaphysis and stabilized with a mini-plate. A gelfoam piece impregnated with a solution of EPCs (1x106) was placed into the fracture gap. Control animals received only saline-gelfoam with no cells. In total, 42 rats were studied: 21 in EPC and 21 in control groups. Seven animals were sacrificed from each group at one, two, and three weeks post-operatively. Plain radiographs of the operated femur were taken before sacrifice. Operated femurs were harvested and the specimens from the osteotomy site were collected for histological evaluation. The x-rays were scored in a scale from zero to five according to the percentage and the intensity of the bone filling at the osteotomy site. Hematoxylin-eosin stained slides were evaluated for new vessel formation and the amount of bone tissue.

Results: Radiographically, at three weeks, the mean score for the EPC group was 4.5 with five out of seven animals having bridging callus; whereas for the control group, the mean score was 2.2 with no bridging callus formation. At two weeks, EPC treated animals had a mean score of 2.4, and the control group had a score of 1. Bone formation was insignificant at one week in either group, however, the scores tended to be higher in the EPC group animals than the control; 0.6 to 0.3 respectively. Histological evaluation revealed that the specimens from EPC treated animals had abundant spicules of trabecular bone containing predominantly bone cells, osteoid, and new vessels. Conversely, control animals had scarce trabecular bone with markedly less bone cells and vessels.

Conclusion: Local EPC therapy stimulates angiogenesis and increases osteogenesis and callus formation post fracture. Our report encourages further investigation of the local use of EPCs as a potential therapy to promote bone regeneration.

Purpose: Micro-CT is efficient, non-destructive, and accurate for qualitative and quantitative studies of bone microarchitecture during fracture healing. A cell-based vascular endothelial growth factor (VEGF) gene delivery system can increase fracture healing. Three dimensional structural variation of new bone formation in rabbit fracture segmental defects was studied with micro-CT to determine how VEGF affects these microarchitectural differences for bone healing in various periods.

Method: All animal procedures were approved by the Animal Care Committee at St. Michael’s hospital. Ten millimeter segmental bone defects were treated by local injection with cell-based VEGF gene transfer (n=15), or control group with fibroblasts alone or saline only (n=15), to stimulate differences in bone healing. The animals were sacrificed and fracture healing specimens collected at 4, 8 and 12 weeks post surgery. The region of interest (ROI) was set where the segmental defect was located, and was selected for analysis from the recognizable margins of the original defect. To describe the topographic pattern of bone healing, the ROI was divided into three areas of equal volume: proximal, middle and distal. The new bone formation and mineralization at the defect sites were evaluated by bone structural parameters from the 3-D reconstruction of micro-CT.

Results: Macroscopic evaluation of the interfragmentary section from reconstructed micro CT scans, in the VEGF treated rabbits, showed abundant fragmentary bone filling the gap of the osteotomy at 4 weeks and abundant callus bridging the gap at 8 and 12 weeks. In the control group, only small amounts of sparsely formed bone were seen in the gap at 4 weeks. In the control group, the regenerate bone was ovoid around the bone sites and a big gap remained in the segmental bone defects at 8 and 12 weeks. The bone healing micro-structural differences between the two groups varied with the period of treatment, with more differences seen at 4 than 8 or 12 weeks.

Conclusion: Cell-based VEGF gene therapy enhances fracture healing of segmental defects, and this effect is best seen in the early period following defect creation.

Aim of the study: To evaluate the use of a gelfoam sponge as a scaffold material in delivering osteoblast cells transfected with the VEGF gene for fracture repair.

Methods: In vitro: Osteoblasts were cultured from periosteum of rabbit bone and labeled with the visible CMTMR. Commercially available gelfoam with 12 pieces (each 3 × 3 × 3 mm3) was impregnated and cultured with the labelled cells (1×106) in a 12 wells plate for 1, 3 and 7 days. We embedded the gelfoam with labeled cells in an OCT compound enface, and the sections were then examined under a fluorescent microscope. In vivo: Osteoblasts were transfected with VEGF by use of SuperFect (Qiagen Inc) and cultured for 24 hours. The gelfoam pieces were impregnated with the transfected cells (5×106) saline solution for 30 minutes and placed into a segmental bone defect created in the rabbit tibia for 7 (n=3) and 14 (n=3) days. The specimens including the new bone were cut through each site of the segmental defect and embedded in paraffin. The sections were dewaxed and immunostained with mouse anti-human VEGF.

Results: In vitro: CMTMR-labeled cells survived and were detected within gelfoam at different time intervals (days 1, 3 and 7). In vivo: Immunostained VEGF proteins were visualized in the tissues surrounding the residual gel-foam at the fracture site at days 7 and 14 post surgery.

Conclusion: Our results indicate that the labeled/transfected cells are capable of growth in a gelfoam sponge both in vitro and in vivo.

Particulate wear debris from the UHMWPE component of implant prostheses typically causes inflammatory cascades leading to bone resorption and prosthesis loosening. Aseptic loosening is the leading cause of joint replacement failure. Green et al. have shown that the most biologically active polyethylene wear particles are in size range 0.3–10 micrometer, determined by filtration and Scanning Electron Microscopy.

A new methodology based on radioisotope tracing is investigated which promises aseptic loosening is the leading cause of joint replacement failureto be more sensitive and may allow the characterization of wear debris shedding on the nanometer-scale. A constant force knee simulator has been designed and constructed at the University of New South Wales, to generate reproducible wear patterns. Atomic Force Microscopy is used to measure the wear particle dimensions.

The constant axial force can be adjusted over a range of 0–1000 N, and flexion angles of 24°, 38°, 51° and 66° can be set. The UHMWPE wear surface is articulated at a rate of 1 cycle per second. It has been found that the simulator operates reliably over up to 2×10^6 cycles at various loads and flexion angles, and that wear debris can successfully be removed from the lubricant. For a walking cycle simulation, a wear rate of the order of 86 mg/10^6 cycles was measured using distilled water as lubricant.

The debris particulates generated from the simulation have been characterized with Atomic Force Microscopy. In the nanometer range two characteristic types, clumps and fibrils, may be distinguished.

A constant force knee simulator has been shown to operate reliably over up to 2×10^6 cycles at various loads and flexion angles, and that wear debris particulates can be obtained. It has also been shown that atomic force microscopy is well suited to characterize nanometre size UHMWPE particles. In parallel, the wear debris generated from the experiments is being tested for their bioirritant characteristics on osteoblast cells (in the TORU laboratory at the John Curtin School of Medical Research at ANU).

Purpose: Vascular Endothelial Growth Factor (VEGF) is vital for both angiogenesis and osteogenesis. The aim of this study was to investigate the effect of cell based VEGF gene delivery on the proliferation and mineralization of rabbit osteoblasts in vitro.

Method: Primary cultured rabbit osteoblasts were divided into four groups (each n=6). In Group I, osteoblasts were transfected with pcDNA3.1-VEGF; in Group II, osteoblasts were transfected with pcDNA-Efficiency Green Fluorescent Protein (EGFP); in Group III, osteoblasts were treated with the supernatant of fibroblasts that were transfected with VEGF genes; and in Group IV, osteoblasts were treated with the supernatant of fibroblasts that were transfected with EGFP. The cells were cultured in a-EME with 10% FBS, 2% penicillin/streptomycin with or without 10-^7 M dexamethasone and 50μg/ml L-ascorbic acid for 28 days. In the last 4 days, the cells were stimulated to initiate calcium mineralized nodule formation by adding 10 mM B-glycerophosphate. They were stained by the Von Kossa technique so that the number and the area of the nodules could be assessed by an imaging analysis system.

Results: The cells transfected by VEGF were indicated by the EGFP marked cells under a fluorescent microscope. There was a significant difference in the total nodule area (mean 18.38 mm² SE 3.73 and 5.07 mm² SE 0.55, p< 0.05) and count (mean 18.67 SE 3.22 and 2.17 SE 0.40, p< 0.001) between Group I and Group II (ANOVA, SPSS). More unmineralized and smaller nodules were found in Group III and Group IV. However, the nodules in Group III covered greater areas with dark brown staining in the cell culture dishes when compared with Group IV.

Conclusion: The observations indicate that cell based VEGF gene delivery has a positive effect on the proliferation and mineralization of osteoblasts. The greatest effect is seen with direct transfection of osteoblast cells. Cell-based VEGF gene therapy may be used to promote fracture healing.

The purpose of this study was to develop a cell-based VEGF gene therapy in order to accelerate fracture healing and investigate the effect of VEGF on bone repair in vivo.

Twenty-one rabbits were studied. A ten millimeter segmental bone defect was created after twelve millimeter periosteal excision in the middle one third of each tibia and each tibia was plated. Primary cultured rabbit fibroblasts were transfected by use of SuperFect (Qiagen Inc) with pcDNA-VEGF. 5.0 X 106 cells in 1ml PBS were delivered via impregnated gelfoam into the fracture site. Experimental groups were:

Transfected fibroblasts with VEGF (n=7),

Radiology: Fracture healing was defined as those with bone bridging of the fracture defect. After ten weeks, fourteen tibial fractures were healed in total including six in group one, four in group two and four in group three. The VEGF group had an earlier initial sufficient volume of bridging new bone formation. Histological evaluation demonstrated ossification across the entire defect in response to the VEGF gene therapy, whereas the defects were predominantly fibrotic and sparsely ossified in groups two and three. Numerous positively stained (CD31) vessels were shown in the VEGF group. MicroCT evaluation showed complete bridging for the VEGF group, but incomplete healing for groups two and three. Micro-CT evaluation of the new bone structural parameters showed that the amount of new bone (volume of bone (VolB) x bone mineral density (BMD)), bone volume fractions (BVF), bone volume/tissues (BV/TV), trabecular thickness (Tb.Th), number (Tb.N) and connectivity density (Euler number) were higher; while structure model index (SMI), bone surface/bone volume (BS/BV), and trabecular separations (Tb.Sp) were lower in the VEGF group than the other groups. P-Values < 0.05 indicated statistical significance (ANOVA, SPSS) in all parameters except for SMI (0.089) and VolBx-BMD (0.197).

These results indicate that cell-based VEGF gene delivery has significant osteogenic and angiogenic effects and demonstrates the ability of cell based VEGF gene therapy to enhance healing of a critical sized defect in a long bone in rabbits.

Fracture healing continues to pose challenges for researchers and clinicians in the field of trauma and orthopaedic surgery. The future treatment strategies for fracture healing will most likely focus on the use of biologic and biochemical methods in combination with established fixation and mechanical methods. In this study, heparanase (HPSE), a mammalian endo-glycuronidase that promotes angiogenesis through cleavage of the extra cellular matrix (ECM)-heparan sulphate and mobilization of ECM resident growth factors, was investigated for its osteoblasts-stimulating effect.

Osteoblast cells, originated from osteoporotic and healthy human subjects who underwent total knee replacement, were cultured and exposed to HPSE at a series of final concentrations of 1, 3, and 6μg/mL. The cell density, proliferation, alkaline phosphatase (ALP) production and specific activity, and expression of osteogenic genes were examined.

A marked stimulating effect of HPSE in cell density and proliferation was observed in the osteoblastic cultures from both osteoporotic and healthy subjects. The ALP level and its specific activity, a classical osteoblastic marker, were also increased at the presence of HPSE in a dose-dependant manner. The expression of osteogenic pathway genes, particularly bone morphogenic proteins (BMPs), transcription factors SMDs, vascular endothelial growth factor and tissue inhibitor of metallopeptidase (TIMP) were up- or down-regulated, which correlated with the doses of HPSE.

This study is the first to show that HPSE increases cell proliferation and stimulates differentiation in human osteoblasts suggesting that the potential of HPSE as a new biofactor for the treatment of fractures. Further research on HPSE in co-culture of osteoblasts and osteoclasts is under investigation in our laboratory.

We sought to establish whether fibroblasts transfected ex vivo could be delivered via gelfoam impregnated with a solution of transfected cells to achieve local transgene expression in a fracture site.

A 10 millimeter segmental bone defect was created after 12 mm periosteal excision and plated in the middle one third of each rabbit tibia. Dermal tissues were obtained and fibroblasts were cultured with DMEM. Fibroblasts were labeled with CMTMR and 5x106 labeled fibroblasts in 1ml PBS with 1x1 cm? Impregnated gelfoam was placed into the fracture gap (n=2). Twenty four hours after cell injection, the rabbits were killed and specimens were harvested from the fractured leg. Using SuperFect (Qiagen Inc), the primary fibroblasts were transfected with pcDNA-VEGF which was generated with the full length coding sequence of the human VEGF gene. A convenient reporter gene, Efficiency Green Fluorescent Protein (EGFP), was used for monitoring transfection of VEGF by fluorescence intensity. Experimental rabbits received 5.0 X 106 VEGF transfected cells in 1 ml PBS via gelfoam at the fracture sites. The animals were sacrificed at seven days (n=4), fourteen days (n=4) and twenty-one days (n=4) post surgery and the fracture site specimens were collected for analysis.

The fluorescently labeled cells with CMTMR were found at the fracture site and surrounding tissues. It was demonstrated that the labeled cells were delivered into the fracture gap, bone marrow and muscle surrounding a segmental defect in the rabbit. In the VEGF group, visualised VEGF immunostaining (brown) was shown in the fracture site around the Gelfoam; as well VEGF was distributed at sites of endochondral ossification. Visible bone formation was shown: VEGF promoted new bone formation by VonKossa staining (dark) and produced numerous vessels by CD31 positive staining (brownish black). The VEGF protein was detected in and around the fracture by ELISA.

This data encourages the further development of genetic approaches using cell based VEGF gene transfer without viral vectors to promote fracture healing.