Additive Influence of Extracellular pH, Oxygen Tension, and Pressure on Invasiveness and Survival of Human Osteosarcoma Cells.

BACKGROUND/PURPOSE: The effects of chemical and physical interactions in the microenvironment of solid tumors have not been fully elucidated. We hypothesized that acidosis, hypoxia, and elevated interstitial fluid pressure (eIFP) have additive effects on tumor cell biology and lead to more aggressive behavior during tumor progression. We investigated this phenomenon using three human osteosarcoma (OS) cell lines and a novel in vitro cell culture apparatus. MATERIALS AND METHODS: U2OS, SaOS, and MG63 cell lines were cultured in media adjusted to various pH levels, oxygen tension (hypoxia 2% O2, normoxia 20% O2), and hydrostatic gage pressure (0 or 50 mmHg). Growth rate, apoptosis, cell cycle parameters, and expression of mRNA for proteins associated with invasiveness and tumor microenvironment (CA IX, VEGF-A, HIF-1A, MMP-9, and TIMP-2) were analyzed. Levels of CA IX, HIF-1α, and MMP-9 were measured using immunofluorescence. The effect of pH on invasiveness was evaluated in a Matrigel chamber assay. RESULTS: Within the acidic-hypoxic-pressurized conditions that simulate the microenvironment at a tumor's center, invasive genes were upregulated, but the cell cycle was downregulated. The combined influence of acidosis, hypoxia, and IFP promoted invasiveness and angiogenesis to a greater extent than did pH, pO2, or eIFP individually. Significant cell death after brief exposure to acidic conditions occurred in each cell line during acclimation to acidic media, while prolonged exposure to acidic media resulted in reduced cell death. Furthermore, 48-h exposure to acidic conditions promoted tumor invasiveness in the Matrigel assay. CONCLUSION: Our findings demonstrate that tumor microenvironmental parameters - particularly pH, pO2, and eIFP - additively influence tumor proliferation, invasion, metabolism, and viability to enhance cell survival and must be controlled in OS research.

Tumour interstitial fluid pressure may regulate angiogenic factors in osteosarcoma.

PURPOSE: We have previously shown that osteosarcomas have states of increased interstitial fluid pressure (IFP) which correlate with increased proliferation and chemosensitivity. In this study, we hypothesized that constitutively raised IFP in osteosarcomas regulates angiogenesis. MATERIALS AND METHODS: Sixteen patients with the clinical diagnosis of osteosarcomas underwent blood fl ow and IFP readings by the wick-in-needle method at the time and location of open biopsy. Vascularity was determined by capillary density in the biopsy specimens. We performed digital image analysis of immunohistochemical staining for CD31, VEGF-A, VEGF-C and TPA on paraffin-embedded tissue blocks of the biopsy samples. Clinical results were validated in a pressurised cell culture system. RESULTS: IFPs in the tumours (mean 33.5 +/- SD 17.2 mmHg) were significantly higher (P = 0.00001) than that in normal tissue (2.9 +/- 5.7 mmHg). Pressure readings were significantly higher in low vascularity tumours compared to high vascularity tumours (P <0.001). In the osteosarcoma cell lines, growth in a pressurised environment was associated with VEGF-A downregulation, VEGF-C upregulation and TPA upregulation. The reverse was seen in the OB cell lines. Growth in the HUVEC cell line was not significantly inhibited in a pressurised environment. Immunohistochemical assessment for VEGF-A (P = 0.01), VEGF-C (P = 0.008) and TPA (P = 0.0001) translation were consistent with the findings on PCR. CONCLUSION: Our data suggest that some molecules in angiogenesis are regulated by changes in IFP.

Tumor interstitial fluid pressure may regulate angiogenic factors in osteosarcoma.

We have previously shown that osteosarcomas (OS) have states of increased interstitial fluid pressure (IFP), which correlate with increased proliferation and chemosensitivity. In this study, we hypothesized that constitutively raised IFP in OS regulates angiogenesis. Sixteen patients with the clinical diagnosis of OS underwent blood flow and IFP readings by the wick-in-needle method at the time and location of open biopsy. Vascularity was determined by capillary density in the biopsy specimens. We performed digital image analysis of immunohistochemical staining for CD31, VEGF-A, VEGF-C, and TPA on paraffin-embedded tissue blocks of the biopsy samples. Clinical results were validated in a pressurized cell culture system. Interstitial fluid pressures in the tumors (mean 33.5 +/- SD 17.2 mmHg) were significantly higher (p = 0.00001) than that in normal tissue (2.9 +/- 5.7 mmHg). Pressure readings were significantly higher in low vascularity tumors compared to high vascularity tumors (p < 0.001). In the OS cell lines, growth in a pressurized environment was associated with VEGF-A downregulation, VEGF-C upregulation, and TPA upregulation. The reverse was seen in the OB cell line. Growth in the HUVEC cell line was not significantly inhibited in a pressurized environment. Immunohistochemical assessment for VEGF-A (p = 0.01), VEGF-C (p = 0.008), and TPA (p = 0.0001) translation were consistent with the findings on PCR. Our data suggests that some molecules in angiogenesis are regulated by changes in IFP.

Bisphosphonate delivery to tubular bone allografts.

Large structural allografts used for reconstruction of bone defects after revision arthroplasty and tumor resection fracture up to 27% of the time from osteolytic resorption around the fixation screw holes and tendon or ligament attachment sites. Treating structural allografts before implantation with bisphosphonates may inhibit local osteoclastic processes and prevent bone resorption and the development of stress risers, thereby reducing the long-term fracture rate. Taking advantage of allografts' open-pore structure, we asked whether passive soaking or positive-pressure pumping was a more efficient technique for delivering bisphosphonates. We treated matched pairs of ovine tibial allografts with fluids containing Tc-99m pamidronate and toluidine blue stain to facilitate indicator distribution analysis via microSPECT-microCT imaging and light microscopy, respectively. Surfactants octylphenoxy polyethoxy ethanol or beractant were added to the treatment fluids to reduce flow resistance of solutions pumped through the allografts. Indicator distribution after 1 hour of soaking produced a thin ring around periosteal and endosteal surfaces, while pumping for 10 minutes produced a more even distribution throughout the allograft. Flow resistance was reduced with octylphenoxy polyethoxy ethanol but unaffected with beractant. Pumped allografts displayed a more homogeneous indicator distribution in less time than soaking while surfactants enhanced fluid movement.

Influence of carboplatin infusion on osteosarcoma blood flow.

PURPOSE: Herein we report that carboplatin infusion influenced tumor blood flow signal independent of the mechanical decompression induced by the artificial lymphatics system technology that was being evaluated as part of a randomized veterinary clinical trial, treating spontaneously occurring canine appendicular osteosarcoma, a tumor very similar to its human counterpart. METHODS: Blood flow within the central region of the tumor was recorded continuously using laser Doppler flowmetry, a real-time measurement technology. Time-averaged flow values were computed from segments taken from the recordings immediately before starting carboplatin infusion, and during infusion. RESULTS: Carboplatin increased the tumor blood flow signal by an additional 59 +/- 26% (mean +/- SEM; p = 0.06) over the increase induced by the decompression. The increase started within 49 +/- 46 s after the start of infusion, had a response time constant of 19 +/- 21 s and persisted throughout the infusion, ending shortly after infusion ended. CONCLUSION: The rapidity of the flow signal increase suggests that carboplatin may have an autonomic effect on circulation, either local or systemic. The observations identify a new action of this drug and suggest a possible mechanism to exploit therapeutically.

Use of an artificial lymphatic system during carboplatin infusion to improve canine osteosarcoma blood flow and clinical response.

BACKGROUND: The artificial lymphatic system (ALS), a mechanical system designed to reduce increased interstitial fluid pressure in solid tumors and enhance the delivery of chemotherapy, was evaluated within a randomized clinical trial treating spontaneously occurring canine appendicular osteosarcoma (OS), a tumor similar to its human OS counterpart. METHODS: An ALS was investigated for its ability to increase OS blood flow and increase uptake of intravenously administered carboplatin. RESULTS: Blood flow increased by 314% in tumors with active ALS drains versus 126% in control tumors (P < .03). Tumor carboplatin uptake increased by 51% after drain activation (P = .07). Microvascular density (MVD) was measured in tumors after surgical amputation and in corresponding bone regions in a cohort of normal dogs. The OS tumors had equivalent MVD as normal bone, and MVD was higher in the humerus than the femur (P < .03) in both tumor and normal bone. Median survival between the ALS-treated and control cohorts was not different despite increased drug uptake or ALS manipulation. Compared with historic controls, ALS drain insertion into tumors to reduce interstitial fluid pressure did not worsen the prognosis. CONCLUSIONS: The findings in canine spontaneously occurring OS indicate that an ALS may be of value as a chemotherapy adjunct for enhancing the delivery of chemotherapy to tumor interstitium.

Use of an absorbable membrane to position biologically inductive materials in the periprosthetic space of cemented joints.

A device is presented that positions ultrahigh molecular weight polyethylene (UHMWPE) debris against periprosthetic bone surfaces. This can facilitate the study of aseptic loosening associated with cemented joint prostheses by speeding the appearance of this debris within the periprosthetic space. The device, composed of a 100 microm thick bioabsorbable membrane impregnated with 1.4 x 10(9) sub-micron particles of UHMWPE debris, is positioned on the endosteum of the bone prior to the insertion of the cemented orthopedic implant. An in vitro pullout study and an in vivo canine pilot study were performed to investigate its potential to accelerate "time to aseptic loosening" of cemented prosthetic joints. Pullout studies characterized the influence of the membrane on initial implant fixation. The tensile stresses (mean+/-std.dev.) required to withdraw a prosthesis cemented into canine femurs with and without the membrane were 1.15+/-0.3 and 1.54+/-0.01 MPa, respectively; these findings were not significantly different (p > 0.4). The in vivo pilot study, involving five dogs, was performed to evaluate the efficacy of the debris to accelerate loosening in a canine cemented hip arthroplasty. Aseptic loosening and lameness occurred within 12 months, quicker than the 30 months reported in a retrospective clinical review of canine hip arthroplasty.

Cell proliferation of cultured human cancer cells are affected by the elevated tumor pressures that exist in vivo.

Elevated interstitial fluid pressure (IFP) is observed in most solid tumors. However, the study of the cellular processes of tumors and the development of chemotherapy are routinely studied using in vitro culture systems at atmospheric pressure. Using a new pressurized cell culture system, we investigated the influence of hydrostatic pressure on population dynamics of three primary osteosarcoma (HOS, U2OS, SaOS2) and two metastatic tumor cell lines (MCF7 breast, H1299 lung) that invade bone. Values of IFP in normal human bone and muscle, and in osteosarcoma tumors obtained during their surgical biopsy established the hydrostatic pressure range for the in vitro cell studies. The IFP values were obtained from a retrospective review of patient records. IFP from confirmed osteosarcoma was 35.9+/- 16.2 mmHg. Tumor IFP was significantly higher than muscle IFP (p < 0.001) and bone IFP (p < 0.003). The in vitro study measured the cell-line proliferation using hydrostatic pressures of 0, 20, 50 and 100 mmHg. The findings suggest that hydrostatic pressure either increases or decreases tumor proliferation rates depending on cell type. Furthermore, cell death was not associated with apoptosis.

Elevated physiologic tumor pressure promotes proliferation and chemosensitivity in human osteosarcoma.

PURPOSE: This study investigates the effect of constitutively raised interstitial fluid pressure on osteosarcoma physiology and chemosensitivity. EXPERIMENTAL DESIGN: We did pressure and blood flow assessments at the time of open biopsy in patients with the diagnosis of high-grade osteosarcoma and correlated this to survival and chemotherapy-associated tumor necrosis. Osteosarcoma cell lines were then evaluated for proliferative and therapeutic indices in a replicated high-pressure environment. RESULTS: Sixteen osteosarcomas in vivo were assessed and exhibited elevated interstitial fluid pressures (mean 35.2 +/- SD, 18.6 mmHg). This was not associated with significantly impeded blood flow as measured by a Doppler probe at a single site (P < 0.12). Nonetheless, greater chemotherapy-associated necrosis and associated longer survival were seen in tumors with higher interstitial fluid pressures (P < 0.05). In vitro, cells undergo significant physiologic changes under pressure. Osteosarcoma cell lines grown in a novel hydrostatically pressurized system had variable cell line-specific growth proportional to the level of pressure. They were more proliferative as indicated by cell cycle analysis with more cells in S phase after 48 hours of pressurization (P < 0.01). There was a significant elevation in the cell cycle-related transcription factors E2F-1 (P < 0.03) and E2F-4 (P < 0.002). These changes were associated with increased chemosensitivity. Cells tested under pressure showed an increased sensitivity to cisplatin (P < 0.00006) and doxorubicin (P < 0.03) reminiscent of the increased chemotherapy-associated necrosis seen in tumors with higher interstitial fluid pressure in the clinical study. CONCLUSIONS: The results of this study suggest that cells in the in vivo pressurized environment are at a higher state of regenerative activity than is demonstrable in conventional cell culture systems. Variations in tumor interstitial fluid pressure have the potential to alter chemotherapeutic effects.

Dog osteogenic sarcoma microvasculature observed with a Spälteholz technique.

A well-distributed, patent microvascular network is essential for adequate, uniform delivery of chemotherapy into solid tumors. This network has not been evaluated in osteogenic sarcoma. Spälteholz tissue clarification was used to observe the microvasculature of canine humeri bearing osteogenic sarcoma. Freshly amputated limbs, obtained from therapeutic amputation, were infused with a micron-sized carbon particle solution, frozen, and then cut into sagittal and axial 0.5-mm thick sections. They were photographed, then radiographed using high resolution Faxitron xray, chemically treated to clarify the tissue, and then rephotographed. Microvasculature was identified by the localization of carbon particles, which were unaffected by the clarification process, within the clarified sections. Clarified section photographs were digitized to gray scale levels and analyzed using IMAGE software; levels are directly related to capillary density. Faxitron and original images were registered to the clarified images to identify tissue regions. Multiple regions of interest from normal muscle, fat, bone, and tumor regions were selected and averaged. The microvasculature of the tumor was inhomogeneous, whereas its density was considerably lower than normal adjacent muscle and bone (range, 56-72% lower). These findings suggest that insufficient microvascular density and distribution may provide additional explanation for the poor response of solid tumors to chemotherapy and radiation therapy.

Biomechanical analysis of anterior poly-methyl-methacrylate reconstruction following total spondylectomy for metastatic disease.

STUDY DESIGN: Three reconstruction options were evaluated biomechanically following total spondylectomy using human cadaveric spine specimens. OBJECTIVES.: To evaluate and compare the stability of combined anterior and posterior fixation incorporating poly-methyl-methacrylate with alternative accepted reconstruction techniques. SUMMARY OF BACKGROUND DATA: Total spondylectomy represents the most radical option for decompression in metastatic spinal cord compression. Poly-methyl-methacrylate is considered a useful adjunct in spinal column stabilization and arthrodesis; however, there is little published biomechanical data to support its use in this setting. METHODS: Ten fresh-frozen human cadaveric spines (T9-L3) were used. After intact analysis, a total spondylectomy was performed at T12. Three potential reconstruction techniques were tested for their ability to restore stiffness to the specimen: 1) multilevel posterior pedicle screw instrumentation from T10-L2; 2) anterior instrumentation (ATL Z plate II) and rib graft at T11-L1 with multilevel posterior pedicle screw instrumentation from T10-L2; and 3) anterior cement (Simplex P) and pins construct (T12) with multilevel posterior pedicle screw instrumentation from T10-L2. Each of the three potential reconstruction techniques was tested on each specimen in random order using nondestructive testing under load control. RESULTS: Only combined stabilization techniques (e.g., anterior instrumentation and rib graft with multilevel posterior pedicle screw instrumentation and anterior cement-and-pins construct with multilevel posterior pedicle screw instrumentation) restored stiffness to a level equivalent to or higher than that of the intact spine in all loading modes (P < 0.05). Anterior cement-and-pins construct with multilevel posterior pedicle screw instrumentation provided more stability to the specimen than anterior instrumentation and rib graft with multilevel posterior pedicle screw instrumentation in compression and flexion testing (P < 0.05). Posterior instrumentation alone did not restore stiffness to the intact level in compression and flexion testing (P < 0.005). CONCLUSIONS: Combined anterior and posterior reconstruction using a cement construct provides equal to or more stability than the intact spine in all testing modes. Posterior stabilization alone is an inferior method of reconstruction following total spondylectomy. Poly-methyl-methacrylate has the advantage over traditional anterior reconstruction techniques in that it can be inserted using a posterior approach.

PMMA to stabilize bone and deliver antineoplastic and antiresorptive agents.

Antineoplastic and antiresorptive drugs added to polymethylmethacrylate cement may prevent local cancer progression and failure of reconstructive devices used to treat patients with pathologic fractures. We tested the mechanical properties of cement containing various amounts of the drugs and found that as much as 2 g of either doxorubicin or pamidronate can be added to Simplex cement and the cement retains 87% of its compressive and tensile strength after 6 months of wet storage. Approximately 1 mg pamidronate elutes from experimental pellets. One half of the drug elution occurs within the first day in experiments that combined doxorubicin and pamidronate, and within 3 days when pamidronate was the only additive. Cement containing these drugs seems to be strong enough, but its fatigue strength should be tested before using it clinically. Sufficient amounts of the tested drugs elute to have potential biologic activity.