2 and IGF-1 respectively enhanced proliferation and viability most effectively, increasing these two parameters in particle-treated cultures by 30-50% more than those not exposed to growth factors, and at levels beyond the control. FGF-2, IGF-1, and BMP-6 enhanced expression of type I collagen, alkaline phosphatase, osteocalcin, and BSP in the presence or absence of particles, with BMP-6 being most effective. However, titanium particles in this study had only slightly inhibited proliferation (by 10-15% relative to control), viability (by a non-significant 7%), and expression of collagen type I (38%), alkaline phosphatase (24%), and osteocalcin (15%), while not affecting expression of BSP (Jeong et al., 2008). The study also showed that TGF-1 did not increase alkaline phosphatase, osteocalcin, or BSP expression and was the weakest of the four growth factors in terms of overall trophic effects on hMSCs. In another study, osteogenic protein-1 (OP-1), also known as BMP-7, improved the mineralization and alkaline phosphatase production of MC3T3-E1 pre-osteoblasts treated with PMMA particles. MC3T3-E1 cells exposed to PMMA particles showed a dosedependent decrease in mineralization and alkaline phosphatase production, while the addition of OP-1 at 200 ng/mL to cultures significantly boosted these parameters by 30- 170% at all time periods of treatment (days 1-20, 1-4, or 4-20 out of a 20-day culture period) and all particle doses tested (Kann et al., 2010). This increase in osteogenesis by OP-1 was significant whether particles were added to MC3T3-E1 cultures on the first or fourth day of growth. However, one study showed that the bisphosphonate zoledronate did not improve proliferation of MC3T3-E1 pre-osteoblasts or MG-63 osteoblasts in the presence of titanium particles (at 0.01% wt, the highest dose the cells could endure without showing signs of decreased proliferation relative to controls) throughout a 28 day period and over a wide dose range of zoledronate tested (0.1 to 100 M by increasing orders of magnitude) (Peter et al., 2005).

#### **6.** *In Vitro* **experimental methods**

202 Osteogenesis

Table 2. Summary of orthopaedic particle effects on osteogenic differentiation of osteoprogenitor and marrow stromal cells. Studies are listed in in chronological order. Abbreviations: Ti = titanium particles, UHMWPE = ultrahigh molecular weight polyethylene particles, PMMA = polymethylmethacrylate particles, CoCrMo= cobaltchromium-molybdenum alloy particles, ZrO2 = zirconium oxide particles, hMSC = human marrow stromal cells, mMSC = mouse marrow stromal cells, MC3T3E1 = MC3T3-E1 preosteoblasts. Signs: () indicates decrease, () indicates increase, (+) indicates presence of,

2 and IGF-1 respectively enhanced proliferation and viability most effectively, increasing these two parameters in particle-treated cultures by 30-50% more than those not exposed to growth factors, and at levels beyond the control. FGF-2, IGF-1, and BMP-6 enhanced expression of type I collagen, alkaline phosphatase, osteocalcin, and BSP in the presence or absence of particles, with BMP-6 being most effective. However, titanium particles in this study had only slightly inhibited proliferation (by 10-15% relative to control), viability (by a non-significant 7%), and expression of collagen type I (38%), alkaline phosphatase (24%), and osteocalcin (15%), while not affecting expression of BSP (Jeong et al., 2008). The study also showed that TGF-1 did not increase alkaline phosphatase, osteocalcin, or BSP expression and was the weakest of the four growth factors in terms of overall trophic effects on hMSCs. In another study, osteogenic protein-1 (OP-1), also known as BMP-7, improved the mineralization and alkaline phosphatase production of MC3T3-E1 pre-osteoblasts treated with PMMA particles. MC3T3-E1 cells exposed to PMMA particles showed a dosedependent decrease in mineralization and alkaline phosphatase production, while the addition of OP-1 at 200 ng/mL to cultures significantly boosted these parameters by 30- 170% at all time periods of treatment (days 1-20, 1-4, or 4-20 out of a 20-day culture period) and all particle doses tested (Kann et al., 2010). This increase in osteogenesis by OP-1 was significant whether particles were added to MC3T3-E1 cultures on the first or fourth day of growth. However, one study showed that the bisphosphonate zoledronate did not improve proliferation of MC3T3-E1 pre-osteoblasts or MG-63 osteoblasts in the presence of titanium particles (at 0.01% wt, the highest dose the cells could endure without showing signs of decreased proliferation relative to controls) throughout a 28 day period and over a wide dose range of zoledronate tested (0.1 to 100 M by increasing orders of magnitude)

(-) indicates absence of change.

(Peter et al., 2005).

Most of our current knowledge of osteoblast and osteoprogenitor responses to orthopaedic wear debris comes from in vitro studies. These studies involve at a minimum, purifying and sterilizing particles and characterizing them with respect to size and shape, isolating and expanding cells and treating them with particles, and conducting various outcome measurements with respect to cell proliferation, differentiation, mineralization, viability, gene expression, protein or cytokine production, adhesion, or particle phagocytosis. With respect to gene expression and protein or cytokine production, commonly utilized methods include quantitative PCR after reverse transcription, Northern blot, in situ hybridization, and gene microarray analysis for mRNAs, and ELISA, western blot, immunohistochemistry, and enzyme reaction assays for proteins. Osteoblast proteins such as osteocalcin, bone sialoprotein, osteonectin, and osteopontin are commonly measured by ELISA or western blot. Alkaline phosphatase is commonly measured by enzyme kinetic reaction of cell lysates with para-nitrophenyl phosphate, its substrate, with spectrophotometric quantitation of chromogenic substrate release (p-nitrophenol) at 405 nm, or by immunohistochemistry given it is a cell surface protein. Secreted cytokines such as IL-6, IL-8, RANKL, M-CSF, and MCP-1 are often measured by ELISA of supernatant samples. All of these proteins can be measured by Q-PCR for mRNA expression. Transcription factors such as Runx2, osterix, Dlx5, and Msx2, are often measured by Q-PCR, while their binding to promoter elements in DNA is assessed by mobility shift electrophoresis, which separates protein-DNA complexes on gel. Mobility shift electrophoresis is also used to study the promoter binding activity of NF-B subunits (RelA, NF-B1), the transcription factor regulating particle-induced inflammation, cytokine production, and inhibition of collagen expression. Particle phagocytosis and ensuing morphological and cellular changes can be visualized by confocal, transmission electron, fluorescence, and light or phase contrast microscopy. Intracellular actin networks can be fluorescently visualized by staining with rhodamine or TRITCconjugated phalloidin.

Calcium phosphate mineralization can be visualized by Alizarin Red S or von Kossa staining, measured by radioactive 45Ca incorporation, or by spectrophotometric quantification of o-cresolphthalein complexed with Ca2+ ions extracted from mineral nodules by 0.6 N HCl. Mineralized nodules in culture stained by the von Kossa method (incubation of fixed cultures in 5% silver nitrate under UV light for 30-60 min) can be quantified by NIH Imaging software and expressed as a percentage (total stained area over total culture well area) (Chiu et al., 2009). Mineralized nodules stained by Alizarin Red S can be quantified by spectrophotometrically measuring Alizarin Red dye extracted from mineralized nodules with 10% acetic acid (Gregory et al., 2004). Matrix collagen and proteoglycan content can be measured respectively by radioactive 3H-proline and 35S-sulfate incorporation.

Viability and proliferation are commonly measured outcomes of osteoblasts and osteoprogenitors in particle experiments. A variety of methods are used to assess viability: trypan blue exclusion, MTT assay, fluorescein diacetate uptake, Annexin V stain, LDH (lactate dehydrogenase) assay, and TUNEL or caspase-3 and 7 assays for apoptosis. Proliferation is assessed by cell counting, 3H-thymidine incorporation, BrdU uptake, and Ki67 antigen detection. Commercial sources also have proprietary kits/methods of assessing viability or proliferation (e.g., Alamar Blue, CCK-8 assay). Many of these assays are based

Biological Response of Osteoblasts and Osteoprogenitors to Orthopaedic Wear Debris 205

numerous reports. UHMWPE particles may be sterilized by gamma irradiation from a cesium-137 source, UV light exposure, 70% ethanol wash, or ethylene oxide treatment. Prior membrane fixation in formalin also destroys endotoxin and microbes. To confirm if the retrieved particles are UHMWPE, micro-Raman spectroscopy is used to generate a spectrum of vibrational frequencies from the particles that are matched with those of a UHMWPE standard reference (Wirth et al., 1999; Wolfarth et al., 1997; Visentin et al., 2004). Energy dispersive x-ray analysis provides spectra of elemental composition and can be used to identify metal (titanium, cobalt chromium) and ceramic (aluminium, zirconium) comtaminants in the UHMWPE particle mixture (Affatado et al., 2001; Wolfarth et al., 1997; Maloney et al., 1995). Studies have shown with SEM that UHMWPE wear debris is mostly spherical or round, elongated, oval, or oblong, with rough, grainy surface containing

Particles should be sterilized and confirmed negative of bacterial endotoxin before addition to culture. Endotoxin induces many of the same biologic reactions as wear particles, including inflammation, cytokine production, and osteoclastogenesis, and potentiates the adverse effects of particles (Bi et al., 2001, 2002; Greenfield et al., 2005). Adherent endotoxin has been detected on commercial titanium particles and actual orthopedic implant materials as a result of the manufacturing process, can also originate from distant infections and accumulate on implants at subclinical levels, and are found at higher levels in patients with inflammatory arthritis such as rheumatoid arthritis (Bi et al., 2001; Nalepka et al., 2006; Greenfield et al., 2008). Endotoxins on particles or in supernantant can be measured using commercial assay kits, such as the Limulus Amoebocyte Lysate Kit from BioWhittaker (Walkersville, MD, USA). Endotoxin, if detected on metal particles, can be removed by five or more alternating cycles of incubation in 25% nitric acid at room temperature, then in 0.1 N sodium hydroxide in 95% ethanol at 30 C, each incubation for 18-20 hrs, with PBS

Osteoblasts may be obtained commercially as tumorigenic cell lines or from primary rodent or human sources. Osteoblast cell lines include MG-63, SaOS-2, U-2OS, HOS, OCT-1, and MLO-Y4, among which MG-63 is most popular. MG-63, SaOS-2, U2OS, and HOS cells are osteosarcoma (osteogenic sarcoma) cells derived from human patients, and show osteoblast traits of polygonal morphology, alkaline phosphatase and osteocalcin expression, collagen matrix deposition, and calcium phosphate mineralization. One study has described MG-63 as immature osteoblasts, and OCT-1 as mature secretory osteoblasts derived from the calvaria, and MYO-L4 as osteocytes derived from the long bones, of transgenic mice that express the SV40 T-antigen oncogene driven by the osteocalcin promoter (Lohmann et al., 2002a; Kato et al., 1997). Lineage-specific cell lines can be readily obtained from transgenic mice in which the SV40 T-antigen is driven by the expression of a lineage-specific gene such as that of osteocalcin, which is expressed only in osteoblasts and osteocytes (Bonewald, 1999; Kato et al., 1997). MG-63, SaOS-2, U2OS, and HOS cells are available commercially from American Type Culture Collection (Manassas, VA, USA); primary osteoblasts are also purchasable from certain commercial sources. Primary osteoblasts are often derived from marrow aspirates of human trabecular bone after hip or knee arthroplasty or iliac crest bone after spine fusion, or from calvaria of neonatal rats. Human trabecular or iliac crest bone or neonatal rat calvarium is first minced into < 1 mm3 pieces and then plated in DMEM or - MEM culture medium. Cells migrate out from the minced bone fragments and expand to near confluency in about 2-3 weeks. The minced bone pieces can also be digested in

numerous pits.

washes in between each step (Ragab et al., 1999).

on the ability of viable cells to metabolize substrates (e.g., MTT, fluorescein diacetate, Alamar Blue) into chromogenic or fluorescent products for spectrophotometric measurement, or the ability of dividing cells to incorporate artificial nucleotides (e.g., BrdU, 13H-thymidine) which can then be detected. Other assays are based on the leakage of intracellular proteins during necrosis or cell injury (e.g., LDH), or specific events occurring in apoptosis (e.g., DNA fragmentation, phosphotidylserine inversion, caspase production). Many of these assays are available as commercial kits.

One of the initial steps of in vitro experimentation is obtaining a pure, sterile source of implant particles, either commercially or by isolating them from tissues of patients with failed hip or knee arthroplasties or wear simulator tests. Most studies with titanium, PMMA, or polystyrene particles have obtained these materials from commercial sources, most often Polysciences (Warrington, PA, USA) for PMMA and polystyrene particles, and Alfa Aesar (Ward Hill, MA, USA), now a part of Johnson Matthey, for titanium particles. Polysciences also produces fluorescent (Fluoresbrite) and color-dyed polystyrene particles, while Sigma-Aldrich (St. Louis, MO, USA) produces titanium oxide (rutile) particles. These particles are available in micron and submicron size ranges of phagocytosable size. Several studies have reported use of UHMWPE particles or powder from Hoechst-Celanese (Dallas, TX, USA) or its affiliated company Ticona (Florence, KY, USA), or Stryker-Howmedica-Osteonics (Kalamazoo, MI, USA) in various medical grades, such as GUR 4150. However, the UHMWPE powders obtained from these sources may be predominantly nonphagocytosable (i.e., 20-200 m in size) and must be filtered or subject to sedimentation to isolate particles of smaller size, with subsequent characterization by scanning electron microscopy to validate their actual size range. These particles can be sterilized by washing in 70% ethanol, autoclaving (for metal particles only), overnight UV light exposure, gamma irradiation, or ethylene oxide treatment.

UHMWPE wear debris is more often obtained from granulomatous membrane tissues or synovial fluid collected respectively from failed hip and knee arthroplasties during revision surgery or from serum lubricant in wear simulator tests (Campbell et al., 1995). Membranes are freshly stored in buffered formalin after retrieval, then minced into mm3 sized pieces, washed thoroughly with water, and digested in papain solution (3 mg papain in 10 mL of 50 mM phosphate buffer, pH 6.5, with 2 mM N-acetyl-L-cysteine) at 65 C for 1-3 days (Maloney et al., 1995; Wirth et al., 1999).Insoluble lysates are precipitated by centrifugation; the collected digest supernatant is then sonicated for 10 min and filtrated through a 0.2 µm polyester filter to isolate the UHMWPE particles. The collected particles are washed with water and sterilized in 70% ethanol, then after final washing, stored in DMEM or saline for later addition to cultures (Maloney et al., 1995; Wirth et al., 1999). UHMWPE debris are retrieved from synovial fluid or wear serum by digesting these samples in 5-10 M sodium hydroxide solution for 12-24 hrs at 65 C (Affatato et al., 2001; Chiu et al., 2009; Minoda et al., 2004; Wolfarth et al., 1997). Serum samples may be lyophilized to dryness before digestion (Chiu et al., 2009). The digest supernatant is sonicated, then ultracentrifuged under a layer of 5% sucrose density gradient at high speed for 3 hrs at 4 C. The sucrose solution is collected and ultracentrifuged under a two-layered isopropanol-water density gradient (0.90, 0.96 g/cm3) at high speed for 1 hour. The layer containing UHMWPE debris at the interface of the two volumes of isopropanol is collected, filtered through a 0.1 m polycarbonate filter, and allowed to dry. UHMWPE particles retrieved from patient samples and wear simulator tests are 1.0 m (0.2 to 1.5 m) in size according to SEM analysis in

on the ability of viable cells to metabolize substrates (e.g., MTT, fluorescein diacetate, Alamar Blue) into chromogenic or fluorescent products for spectrophotometric measurement, or the ability of dividing cells to incorporate artificial nucleotides (e.g., BrdU, 13H-thymidine) which can then be detected. Other assays are based on the leakage of intracellular proteins during necrosis or cell injury (e.g., LDH), or specific events occurring in apoptosis (e.g., DNA fragmentation, phosphotidylserine inversion, caspase production).

One of the initial steps of in vitro experimentation is obtaining a pure, sterile source of implant particles, either commercially or by isolating them from tissues of patients with failed hip or knee arthroplasties or wear simulator tests. Most studies with titanium, PMMA, or polystyrene particles have obtained these materials from commercial sources, most often Polysciences (Warrington, PA, USA) for PMMA and polystyrene particles, and Alfa Aesar (Ward Hill, MA, USA), now a part of Johnson Matthey, for titanium particles. Polysciences also produces fluorescent (Fluoresbrite) and color-dyed polystyrene particles, while Sigma-Aldrich (St. Louis, MO, USA) produces titanium oxide (rutile) particles. These particles are available in micron and submicron size ranges of phagocytosable size. Several studies have reported use of UHMWPE particles or powder from Hoechst-Celanese (Dallas, TX, USA) or its affiliated company Ticona (Florence, KY, USA), or Stryker-Howmedica-Osteonics (Kalamazoo, MI, USA) in various medical grades, such as GUR 4150. However, the UHMWPE powders obtained from these sources may be predominantly nonphagocytosable (i.e., 20-200 m in size) and must be filtered or subject to sedimentation to isolate particles of smaller size, with subsequent characterization by scanning electron microscopy to validate their actual size range. These particles can be sterilized by washing in 70% ethanol, autoclaving (for metal particles only), overnight UV light exposure, gamma

UHMWPE wear debris is more often obtained from granulomatous membrane tissues or synovial fluid collected respectively from failed hip and knee arthroplasties during revision surgery or from serum lubricant in wear simulator tests (Campbell et al., 1995). Membranes are freshly stored in buffered formalin after retrieval, then minced into mm3 sized pieces, washed thoroughly with water, and digested in papain solution (3 mg papain in 10 mL of 50 mM phosphate buffer, pH 6.5, with 2 mM N-acetyl-L-cysteine) at 65 C for 1-3 days (Maloney et al., 1995; Wirth et al., 1999).Insoluble lysates are precipitated by centrifugation; the collected digest supernatant is then sonicated for 10 min and filtrated through a 0.2 µm polyester filter to isolate the UHMWPE particles. The collected particles are washed with water and sterilized in 70% ethanol, then after final washing, stored in DMEM or saline for later addition to cultures (Maloney et al., 1995; Wirth et al., 1999). UHMWPE debris are retrieved from synovial fluid or wear serum by digesting these samples in 5-10 M sodium hydroxide solution for 12-24 hrs at 65 C (Affatato et al., 2001; Chiu et al., 2009; Minoda et al., 2004; Wolfarth et al., 1997). Serum samples may be lyophilized to dryness before digestion (Chiu et al., 2009). The digest supernatant is sonicated, then ultracentrifuged under a layer of 5% sucrose density gradient at high speed for 3 hrs at 4 C. The sucrose solution is collected and ultracentrifuged under a two-layered isopropanol-water density gradient (0.90, 0.96 g/cm3) at high speed for 1 hour. The layer containing UHMWPE debris at the interface of the two volumes of isopropanol is collected, filtered through a 0.1 m polycarbonate filter, and allowed to dry. UHMWPE particles retrieved from patient samples and wear simulator tests are 1.0 m (0.2 to 1.5 m) in size according to SEM analysis in

Many of these assays are available as commercial kits.

irradiation, or ethylene oxide treatment.

numerous reports. UHMWPE particles may be sterilized by gamma irradiation from a cesium-137 source, UV light exposure, 70% ethanol wash, or ethylene oxide treatment. Prior membrane fixation in formalin also destroys endotoxin and microbes. To confirm if the retrieved particles are UHMWPE, micro-Raman spectroscopy is used to generate a spectrum of vibrational frequencies from the particles that are matched with those of a UHMWPE standard reference (Wirth et al., 1999; Wolfarth et al., 1997; Visentin et al., 2004). Energy dispersive x-ray analysis provides spectra of elemental composition and can be used to identify metal (titanium, cobalt chromium) and ceramic (aluminium, zirconium) comtaminants in the UHMWPE particle mixture (Affatado et al., 2001; Wolfarth et al., 1997; Maloney et al., 1995). Studies have shown with SEM that UHMWPE wear debris is mostly spherical or round, elongated, oval, or oblong, with rough, grainy surface containing numerous pits.

Particles should be sterilized and confirmed negative of bacterial endotoxin before addition to culture. Endotoxin induces many of the same biologic reactions as wear particles, including inflammation, cytokine production, and osteoclastogenesis, and potentiates the adverse effects of particles (Bi et al., 2001, 2002; Greenfield et al., 2005). Adherent endotoxin has been detected on commercial titanium particles and actual orthopedic implant materials as a result of the manufacturing process, can also originate from distant infections and accumulate on implants at subclinical levels, and are found at higher levels in patients with inflammatory arthritis such as rheumatoid arthritis (Bi et al., 2001; Nalepka et al., 2006; Greenfield et al., 2008). Endotoxins on particles or in supernantant can be measured using commercial assay kits, such as the Limulus Amoebocyte Lysate Kit from BioWhittaker (Walkersville, MD, USA). Endotoxin, if detected on metal particles, can be removed by five or more alternating cycles of incubation in 25% nitric acid at room temperature, then in 0.1 N sodium hydroxide in 95% ethanol at 30 C, each incubation for 18-20 hrs, with PBS washes in between each step (Ragab et al., 1999).

Osteoblasts may be obtained commercially as tumorigenic cell lines or from primary rodent or human sources. Osteoblast cell lines include MG-63, SaOS-2, U-2OS, HOS, OCT-1, and MLO-Y4, among which MG-63 is most popular. MG-63, SaOS-2, U2OS, and HOS cells are osteosarcoma (osteogenic sarcoma) cells derived from human patients, and show osteoblast traits of polygonal morphology, alkaline phosphatase and osteocalcin expression, collagen matrix deposition, and calcium phosphate mineralization. One study has described MG-63 as immature osteoblasts, and OCT-1 as mature secretory osteoblasts derived from the calvaria, and MYO-L4 as osteocytes derived from the long bones, of transgenic mice that express the SV40 T-antigen oncogene driven by the osteocalcin promoter (Lohmann et al., 2002a; Kato et al., 1997). Lineage-specific cell lines can be readily obtained from transgenic mice in which the SV40 T-antigen is driven by the expression of a lineage-specific gene such as that of osteocalcin, which is expressed only in osteoblasts and osteocytes (Bonewald, 1999; Kato et al., 1997). MG-63, SaOS-2, U2OS, and HOS cells are available commercially from American Type Culture Collection (Manassas, VA, USA); primary osteoblasts are also purchasable from certain commercial sources. Primary osteoblasts are often derived from marrow aspirates of human trabecular bone after hip or knee arthroplasty or iliac crest bone after spine fusion, or from calvaria of neonatal rats. Human trabecular or iliac crest bone or neonatal rat calvarium is first minced into < 1 mm3 pieces and then plated in DMEM or - MEM culture medium. Cells migrate out from the minced bone fragments and expand to near confluency in about 2-3 weeks. The minced bone pieces can also be digested in

Biological Response of Osteoblasts and Osteoprogenitors to Orthopaedic Wear Debris 207

(105, 106, 107, 108 particles/mL), and ceramic particles are at times expressed as mass of particles per volume of medium (e.g., 0.1, 1.0, 10.0 mg/mL). Particle doses are also at times

The in vivo response of osteoblasts and osteoprogenitors to orthopaedic wear debris has been studied by our group using two systems: the femoral intramedullary injection model and the bone harvest chamber (BHC). These systems have been used extensively to study tissue reactions to wear debris particles in mouse and rabbit systems, and have been important in helping researchers understand the mechanism of wear particle-induced osteolysis, inflammation, and granulomatosis. The femoral intramedullary injection system allows implant particles suspended in saline or hyaluronan carrier solution to be delivered continuously over a period of weeks into the femoral canal of experimental rodents. The system consists of a mini-osmotic Alzet pump (Durect, Cupertino, CA, USA) that is implanted subcutaneously in the dorsal, interscapular region of the animal. The Alzet pump is connected via tubing passed via a surgically created tunnel in the subcutaneous tissue, to a hollow titanium rod 6 mm, 23 gauge in dimensions. The titanium rod is inserted into the femoral medullary canal through a hole drilled in the intercondylar notch (the groove between the femoral condyles) using needles of increasingly larger diameter (e.g., 27, 25, 23, 21, 19 gauge). The pump holds approximately 250 L of particle suspension, usually at a concentration of 109 to 1010 particles/mL, and infuses particles into the femoral canal at a rate of 0.15-0.25 L/hr over several weeks. The titanium rod localizes particles to the marrow canal and prevents their leakage. After several weeks, the tissue reaction to particles can be evaluated by histology, staining for markers of osteoblasts, such as alkaline phosphatase or osteocalcin, and osteoclasts, such as TRAP (tartrate resistant acid phosphatase) or the vitronectin receptor V3 to visualize on microscope for cell number and density. Alternatively, particles can be injected as a single bolus in a suspension volume ≤ 10 μL, into the tibial medullary canal through a drilled hole in the proximal tibia, as was done in previous studies. The single bolus injection method, however, is less favorable given that it differs from the clinical scenario in which particles are continuously shed from implants over a long period of time; particles injected this way are also not confined as well to the medullary tissue. Local infusion of particles not only allows the study of resident tissue reactions, but also the migratory patterns of luciferase- and GFP-transfected cells injected intravenously into systemic circulation of the animal. Although the femoral intramedullary injection system theoretically allows the behavior of osteoblasts, osteoprogenitors, and multipotent stem cells to be studied in vivo, in practice, this rests upon identifying single, specific markers for these cells to facilitate histological identification. Alkaline phosphatase is not specific enough to identify osteoblasts or to distinguish them from osteoprogenitors or pre-osteoblasts. Multipotent stem cells are currently not defined by a single marker, but by a large panel of complex surface markers. The in vivo study of osteoblasts, osteoprogenitors, and multipotent stem cells, therefore, rests on the discovery of unique markers that allow for easy and accurate identification of

The femoral intramedullary injection system involves an arthrotomy of the knee to access the intercondylar notch, which is accomplished via a quadriceps-patellar approach (Zilber et al., 2008). This approach involves a 5-mm incision on the anterior aspect of the knee; the

expressed as number of particles per cell (e.g., 5, 50, 500, 5000 particles/cell).

**7.** *In Vivo* **experimentation methods**

these cells histologically.

collagenase (1 mg/mL) for 30-60 min or trypsin for 15 min at 37 C prior to plating, to facilitate release of entrapped cells. The osteoblasts should stain positive for alkaline phosphatase, collagen type I, osteocalcin, bone sialoprotein, and calcium phosphate mineral. Osteoprogenitors and multipotent stem cells with osteochondrogenic potential are subpopulations of heterogeneous marrow stromal cells (MSCs) isolated from the medullary cavities of long bones (Peister et al., 2004; Phinney et al., 1999). MSCs are most commonly used to represent osteoprogenitors in experiments involving osteogenic differentiation, as no marker has yet been discovered that specifically identifies and allows purification of pure osteoprogenitors. Human multipotent stem cells capable of differentiating along osteogenic, chondrogenic, and adipogenic lineages are recognized to be positive for CD markers 27, 44, 90, 105, and 166, and negative for hematopoietic markers such as CD34, 45, and 14. Multipotent stem cells can be isolated by flow cytometry with these antibodies, or may be purchased as pre-purified stocks from commercial sources. MSCs are normally isolated from mice by injecting culture medium through surgically dissected femurs and tibias using a 20-25 gauge needle with syringe, and plated directly into culture (Chiu et al., 2006, 2007; Peister et al., 2004; Phinney et al., 1999). Non-adherent blood cells are removed with subsequent medium exchange. The adherent MSCs, which include osteoprogenitor and multipotent stem cells, can be expanded in culture or used immediately for experimentation. MSCs can be induced to undergo osteogenic differentiation in DMEM or -MEM containing ascorbic acid or ascorbate-2-phosphate (50 g/mL), dexamethasone (10 nM), and glycerophosphate (10 mM) for 2-3 weeks; a series of studies has also included 1,25-(OH)2D3 (vitamin D3 or calcitriol, 10 nM) (Wang et al., 2002, 2003). Human MSCs are isolated from femoral medullary canals of total hip arthroplasty patients and plated/expanded in culture in the same fashion (Wang et al., 2002, 2003). The mouse pre-osteoblast cell line, MC3T3-E1, is commonly used to study osteogenic differentiation and its transcriptional mechanisms. Several subclones of MC3T3-E1 exist with different mineralization potentials and expression patterns of osteocalcin and bone sialoprotein (Wang et al., 1999). Mineralizing subclones 4 and 14, and non-mineralizing subclones 24 and 30, of MC3T3-E1 are available from American Type Culture Collection. All subclones of MC3T3-E1 produce collagenous extracellular matrix, are responsive to ascorbic acid, and express alkaline phosphatase and Runx2/Cbfa1 (Choi et al., 1996; Franceschi et al., 1992, 1994; Quarles et al., 1992; Torii et al., 1996; Xiao et al., 1997, 2002).

Osteoblasts, osteoprogenitors, and MSCs are usually treated with particles in 6-, 12-, or 24 well plates, and occasionally in 96-well plates for proliferation and viability assays. Cells are generally plated at an initial density of 0.5-5.0 x 104 cells/cm2, and then treated with particles after 24 hrs of plating or after reaching 70-80% confluency. hMSCs are usually plated at lower cell densities of 0.5-1.0 x 104 cells/cm2, because of their slow proliferation potential and greater difficulty in expanding these cells to large numbers, compared to osteoblast cell lines which proliferate very robustly. Studies have represented particle doses as weight or volume percentages (relative to culture medium volume), number of particles per cell, number of particles per volume of medium, or mass of particles per well or volume of medium. Units can be interconverted based on knowledge of the average diameter of particles, volume of medium per well, and number of cells per well. PMMA and titanium particles are expressed more often as weight or volume percentages (e.g., 0.1, 0.25, 0.50, 1.0% wt), UHMWPE particles are expressed more often as number of particles per mL of medium (105, 106, 107, 108 particles/mL), and ceramic particles are at times expressed as mass of particles per volume of medium (e.g., 0.1, 1.0, 10.0 mg/mL). Particle doses are also at times expressed as number of particles per cell (e.g., 5, 50, 500, 5000 particles/cell).

#### **7.** *In Vivo* **experimentation methods**

206 Osteogenesis

collagenase (1 mg/mL) for 30-60 min or trypsin for 15 min at 37 C prior to plating, to facilitate release of entrapped cells. The osteoblasts should stain positive for alkaline phosphatase, collagen type I, osteocalcin, bone sialoprotein, and calcium phosphate mineral. Osteoprogenitors and multipotent stem cells with osteochondrogenic potential are subpopulations of heterogeneous marrow stromal cells (MSCs) isolated from the medullary cavities of long bones (Peister et al., 2004; Phinney et al., 1999). MSCs are most commonly used to represent osteoprogenitors in experiments involving osteogenic differentiation, as no marker has yet been discovered that specifically identifies and allows purification of pure osteoprogenitors. Human multipotent stem cells capable of differentiating along osteogenic, chondrogenic, and adipogenic lineages are recognized to be positive for CD markers 27, 44, 90, 105, and 166, and negative for hematopoietic markers such as CD34, 45, and 14. Multipotent stem cells can be isolated by flow cytometry with these antibodies, or may be purchased as pre-purified stocks from commercial sources. MSCs are normally isolated from mice by injecting culture medium through surgically dissected femurs and tibias using a 20-25 gauge needle with syringe, and plated directly into culture (Chiu et al., 2006, 2007; Peister et al., 2004; Phinney et al., 1999). Non-adherent blood cells are removed with subsequent medium exchange. The adherent MSCs, which include osteoprogenitor and multipotent stem cells, can be expanded in culture or used immediately for experimentation. MSCs can be induced to undergo osteogenic differentiation in DMEM or -MEM containing ascorbic acid or ascorbate-2-phosphate (50 g/mL), dexamethasone (10 nM), and glycerophosphate (10 mM) for 2-3 weeks; a series of studies has also included 1,25-(OH)2D3 (vitamin D3 or calcitriol, 10 nM) (Wang et al., 2002, 2003). Human MSCs are isolated from femoral medullary canals of total hip arthroplasty patients and plated/expanded in culture in the same fashion (Wang et al., 2002, 2003). The mouse pre-osteoblast cell line, MC3T3-E1, is commonly used to study osteogenic differentiation and its transcriptional mechanisms. Several subclones of MC3T3-E1 exist with different mineralization potentials and expression patterns of osteocalcin and bone sialoprotein (Wang et al., 1999). Mineralizing subclones 4 and 14, and non-mineralizing subclones 24 and 30, of MC3T3-E1 are available from American Type Culture Collection. All subclones of MC3T3-E1 produce collagenous extracellular matrix, are responsive to ascorbic acid, and express alkaline phosphatase and Runx2/Cbfa1 (Choi et al., 1996; Franceschi et al., 1992, 1994; Quarles et al., 1992; Torii et al.,

Osteoblasts, osteoprogenitors, and MSCs are usually treated with particles in 6-, 12-, or 24 well plates, and occasionally in 96-well plates for proliferation and viability assays. Cells are generally plated at an initial density of 0.5-5.0 x 104 cells/cm2, and then treated with particles after 24 hrs of plating or after reaching 70-80% confluency. hMSCs are usually plated at lower cell densities of 0.5-1.0 x 104 cells/cm2, because of their slow proliferation potential and greater difficulty in expanding these cells to large numbers, compared to osteoblast cell lines which proliferate very robustly. Studies have represented particle doses as weight or volume percentages (relative to culture medium volume), number of particles per cell, number of particles per volume of medium, or mass of particles per well or volume of medium. Units can be interconverted based on knowledge of the average diameter of particles, volume of medium per well, and number of cells per well. PMMA and titanium particles are expressed more often as weight or volume percentages (e.g., 0.1, 0.25, 0.50, 1.0% wt), UHMWPE particles are expressed more often as number of particles per mL of medium

1996; Xiao et al., 1997, 2002).

The in vivo response of osteoblasts and osteoprogenitors to orthopaedic wear debris has been studied by our group using two systems: the femoral intramedullary injection model and the bone harvest chamber (BHC). These systems have been used extensively to study tissue reactions to wear debris particles in mouse and rabbit systems, and have been important in helping researchers understand the mechanism of wear particle-induced osteolysis, inflammation, and granulomatosis. The femoral intramedullary injection system allows implant particles suspended in saline or hyaluronan carrier solution to be delivered continuously over a period of weeks into the femoral canal of experimental rodents. The system consists of a mini-osmotic Alzet pump (Durect, Cupertino, CA, USA) that is implanted subcutaneously in the dorsal, interscapular region of the animal. The Alzet pump is connected via tubing passed via a surgically created tunnel in the subcutaneous tissue, to a hollow titanium rod 6 mm, 23 gauge in dimensions. The titanium rod is inserted into the femoral medullary canal through a hole drilled in the intercondylar notch (the groove between the femoral condyles) using needles of increasingly larger diameter (e.g., 27, 25, 23, 21, 19 gauge). The pump holds approximately 250 L of particle suspension, usually at a concentration of 109 to 1010 particles/mL, and infuses particles into the femoral canal at a rate of 0.15-0.25 L/hr over several weeks. The titanium rod localizes particles to the marrow canal and prevents their leakage. After several weeks, the tissue reaction to particles can be evaluated by histology, staining for markers of osteoblasts, such as alkaline phosphatase or osteocalcin, and osteoclasts, such as TRAP (tartrate resistant acid phosphatase) or the vitronectin receptor V3 to visualize on microscope for cell number and density. Alternatively, particles can be injected as a single bolus in a suspension volume ≤ 10 μL, into the tibial medullary canal through a drilled hole in the proximal tibia, as was done in previous studies. The single bolus injection method, however, is less favorable given that it differs from the clinical scenario in which particles are continuously shed from implants over a long period of time; particles injected this way are also not confined as well to the medullary tissue. Local infusion of particles not only allows the study of resident tissue reactions, but also the migratory patterns of luciferase- and GFP-transfected cells injected intravenously into systemic circulation of the animal. Although the femoral intramedullary injection system theoretically allows the behavior of osteoblasts, osteoprogenitors, and multipotent stem cells to be studied in vivo, in practice, this rests upon identifying single, specific markers for these cells to facilitate histological identification. Alkaline phosphatase is not specific enough to identify osteoblasts or to distinguish them from osteoprogenitors or pre-osteoblasts. Multipotent stem cells are currently not defined by a single marker, but by a large panel of complex surface markers. The in vivo study of osteoblasts, osteoprogenitors, and multipotent stem cells, therefore, rests on the discovery of unique markers that allow for easy and accurate identification of these cells histologically.

The femoral intramedullary injection system involves an arthrotomy of the knee to access the intercondylar notch, which is accomplished via a quadriceps-patellar approach (Zilber et al., 2008). This approach involves a 5-mm incision on the anterior aspect of the knee; the

Biological Response of Osteoblasts and Osteoprogenitors to Orthopaedic Wear Debris 209

solutions (at 595 nm) and referencing the absorbances against a standard curve of turbidities of known particle concentrations. The efficiency was estimated to be 46% for an original load of 6 x 109 polystyrene particles, but it decreased to 23% and then to 15% for approximately each order of magnitude increase in particle number. Efficiency for UHMWPE was approximately one-third for an initial load of 3 x 1010 or 1.5 x 1011 particles (Ortiz et al., 2008a). Particles were successfully pumped into freshly dissected femurs cultured ex vivo in DMEM F-12 medium, and were visible in the medullary canal upon gross inspection, particularly with blue-dyed polystyrene particles (Ortiz et al., 2008b). The efficiency of particle delivery has also been evaluated in live mice (Ma et al., 2008, 2009a). Blue-dued polystyrene particles were injected successfully into the femoral canal of mice over 4 weeks at an efficiency of 40-50% of the initial 6 x 109 particles delivered, and were visible on gross inspection of the dissected femurs (Ma et al., 2009a). UHMWPE particles infused into femurs of mice over 4 weeks led to reduced bone volume and higher numbers of macrophages compared to contralateral control femurs with rod but no particles, as assessed by micro-CT and histology/histomorphometry of femoral cross sections (Ma et al., 2008). These studies established the femoral intramedullary injection model as a successful system for delivering particles in vivo; improvements however, are needed to increase

Another series of studies has used the particle injection method to assess in vivo cell responses to titanium particles (Warme et al., 2004; Epstein et al., 2005b; Bragg et al., 2008). In a similar study, UHMWPE particles injected as a single bolus into the femur of C57BL/6 mice (at an average quantity of 3 x 109 particles/femur in sodium hyaluronate:PBS carrier solution) also induced intramedullary bone marrow monocytes to increase expression of MCP-1, IL-6, and IL-1 over a 10 week period relative to monocytes from control femurs without particles, as determined by RT-PCR of mRNA from extracted monocytes (Epstein et al., 2005a). In the initial study, 1.39 x 108 titanium particles (3.7 ± 1.8 m) were injected as a single bolus in 10 L of sodium hyaluronate: PBS carrier solution into the femoral canal, followed by a press-fit 10 mm, 25 gauge stainless steel rod cut from Kirschner wire to prevent particle leakage (Warme et al., 2004). After 26 weeks, femurs were dissected and cultured ex vivo in DMEM-F12 for 72 hrs. ELISA analysis of culture media revealed that femurs infused with particles yielded a 45, 79, and 221% increase in production of IL-6, MCP-1, and M-CSF compared to contralateral femurs not infused with particles, whereas IL-1 and TNF- levels were not elevated (Warme et al., 2004). Histology of femoral cross sections revealed evidence of endosteal bone scalloping and destruction. In a similar study, the same bolus of titanium particles was injected into femurs of knockout mice lacking IL-1r1, the receptor for IL-1 (B6.129s7-Il1r1, Jackson Laboratories, Bar Harbor, ME, USA) (Epstein et al., 2005b). After 20 weeks, femurs were dissected and placed in organ culture. ELISA analysis of culture medium revealed that the production of MCP-1 by femurs infused with particles in these IL-1r1 knockout mice was no different than contralateral control femurs not infused with particles; the absolute levels of MCP-1 production from these experimental femurs were also significantly lower (6-8 fold lower) than those of wild type mice. However, inflammation and bone loss occurred to similar degrees in both IL-1r1 knockout and wild type mice, which indicated that while lack of the IL-1 receptor limited MCP-1 production, it did not abolish or reduce the overall inflammatory response to particles, due perhaps to activation of alternative inflammatory pathways (Epstein et al., 2005b). A followup study demonstrated histological evidence of inflammation and

efficiency of particle delivery.

Fig. 3. The femoral intramedullary infusion system (dorsal view of mouse). The model consists of an Alzet mini-osmotic pump that is implanted subcutaneously in the interscapular region of the mouse. The pump is connected via tubing to the titanium rod (6 mm, 23 gauge), which is inserted into the medullary canal of the femur via a hole drilled in the intercondylar notch near the knee joint. The tubing is snugged into a surgically created tunnel in subcutaneous tissue. The pump, which holds about 250 L of fluid, is used to infuse particle suspensions into the femoral canal at a rate of 0.15-0.25 L/hour. This model mimics the clinical situation in which bone and marrow tissue are continuously exposed to wear particles produced from implants.

medial patellar ligament is then incised and the patella laterally displaced, which exposes the intercondylar notch for drilling and subsequent insertion of the titanium rod or particle bolus injection. For particle bolus injection, the traditional quadriceps-patellar or the less invasive transpatellar approach can be used (Zilber et al., 2008). In the transpatellar approach, a similar incision is made on the anterolateral side of the knee, the lateral part of the quadriceps is pushed aside, and without cutting the tendons or ligaments or displacing the patella, the intercondylar notch is directly accessed through the patellar tendon for drilling and particle bolus injection. In both approaches, the needle should be drilled 5 mm into the femoral canal to ensure clear passage through the distal femoral growth plate and to prevent leakage. A study has compared the two approaches in mice and shown successful delivery of 10 L volumes of 10% PMMA particles into the femoral canal, confirmed subsequently by micro-CT imaging (Zilber et al., 2008).

The efficiency of this in vivo particle infusion system has been determined in a series of studies (Ortiz et al., 2008a, 2008b; Ma et al., 2008, 2009a). In this series, the Alzet miniosmotic pump delivered UHMWPE and polystyrene particles (0.5 ± 0.015 m) over 4 weeks into an ex vivo collection tube. The pump delivered 109 to 1011 particles suspended in 200 L of mouse serum at a rate of 0.25 L/hr. The efficiency of particle delivery was determined by spectrophotometrically measuring the turbidities of the initial and collected outflow

Fig. 3. The femoral intramedullary infusion system (dorsal view of mouse). The model consists of an Alzet mini-osmotic pump that is implanted subcutaneously in the

interscapular region of the mouse. The pump is connected via tubing to the titanium rod (6 mm, 23 gauge), which is inserted into the medullary canal of the femur via a hole drilled in the intercondylar notch near the knee joint. The tubing is snugged into a surgically created tunnel in subcutaneous tissue. The pump, which holds about 250 L of fluid, is used to infuse particle suspensions into the femoral canal at a rate of 0.15-0.25 L/hour. This model mimics the clinical situation in which bone and marrow tissue are continuously

medial patellar ligament is then incised and the patella laterally displaced, which exposes the intercondylar notch for drilling and subsequent insertion of the titanium rod or particle bolus injection. For particle bolus injection, the traditional quadriceps-patellar or the less invasive transpatellar approach can be used (Zilber et al., 2008). In the transpatellar approach, a similar incision is made on the anterolateral side of the knee, the lateral part of the quadriceps is pushed aside, and without cutting the tendons or ligaments or displacing the patella, the intercondylar notch is directly accessed through the patellar tendon for drilling and particle bolus injection. In both approaches, the needle should be drilled 5 mm into the femoral canal to ensure clear passage through the distal femoral growth plate and to prevent leakage. A study has compared the two approaches in mice and shown successful delivery of 10 L volumes of 10% PMMA particles into the femoral canal, confirmed

The efficiency of this in vivo particle infusion system has been determined in a series of studies (Ortiz et al., 2008a, 2008b; Ma et al., 2008, 2009a). In this series, the Alzet miniosmotic pump delivered UHMWPE and polystyrene particles (0.5 ± 0.015 m) over 4 weeks into an ex vivo collection tube. The pump delivered 109 to 1011 particles suspended in 200 L of mouse serum at a rate of 0.25 L/hr. The efficiency of particle delivery was determined by spectrophotometrically measuring the turbidities of the initial and collected outflow

exposed to wear particles produced from implants.

subsequently by micro-CT imaging (Zilber et al., 2008).

solutions (at 595 nm) and referencing the absorbances against a standard curve of turbidities of known particle concentrations. The efficiency was estimated to be 46% for an original load of 6 x 109 polystyrene particles, but it decreased to 23% and then to 15% for approximately each order of magnitude increase in particle number. Efficiency for UHMWPE was approximately one-third for an initial load of 3 x 1010 or 1.5 x 1011 particles (Ortiz et al., 2008a). Particles were successfully pumped into freshly dissected femurs cultured ex vivo in DMEM F-12 medium, and were visible in the medullary canal upon gross inspection, particularly with blue-dyed polystyrene particles (Ortiz et al., 2008b). The efficiency of particle delivery has also been evaluated in live mice (Ma et al., 2008, 2009a). Blue-dued polystyrene particles were injected successfully into the femoral canal of mice over 4 weeks at an efficiency of 40-50% of the initial 6 x 109 particles delivered, and were visible on gross inspection of the dissected femurs (Ma et al., 2009a). UHMWPE particles infused into femurs of mice over 4 weeks led to reduced bone volume and higher numbers of macrophages compared to contralateral control femurs with rod but no particles, as assessed by micro-CT and histology/histomorphometry of femoral cross sections (Ma et al., 2008). These studies established the femoral intramedullary injection model as a successful system for delivering particles in vivo; improvements however, are needed to increase efficiency of particle delivery.

Another series of studies has used the particle injection method to assess in vivo cell responses to titanium particles (Warme et al., 2004; Epstein et al., 2005b; Bragg et al., 2008). In a similar study, UHMWPE particles injected as a single bolus into the femur of C57BL/6 mice (at an average quantity of 3 x 109 particles/femur in sodium hyaluronate:PBS carrier solution) also induced intramedullary bone marrow monocytes to increase expression of MCP-1, IL-6, and IL-1 over a 10 week period relative to monocytes from control femurs without particles, as determined by RT-PCR of mRNA from extracted monocytes (Epstein et al., 2005a). In the initial study, 1.39 x 108 titanium particles (3.7 ± 1.8 m) were injected as a single bolus in 10 L of sodium hyaluronate: PBS carrier solution into the femoral canal, followed by a press-fit 10 mm, 25 gauge stainless steel rod cut from Kirschner wire to prevent particle leakage (Warme et al., 2004). After 26 weeks, femurs were dissected and cultured ex vivo in DMEM-F12 for 72 hrs. ELISA analysis of culture media revealed that femurs infused with particles yielded a 45, 79, and 221% increase in production of IL-6, MCP-1, and M-CSF compared to contralateral femurs not infused with particles, whereas IL-1 and TNF- levels were not elevated (Warme et al., 2004). Histology of femoral cross sections revealed evidence of endosteal bone scalloping and destruction. In a similar study, the same bolus of titanium particles was injected into femurs of knockout mice lacking IL-1r1, the receptor for IL-1 (B6.129s7-Il1r1, Jackson Laboratories, Bar Harbor, ME, USA) (Epstein et al., 2005b). After 20 weeks, femurs were dissected and placed in organ culture. ELISA analysis of culture medium revealed that the production of MCP-1 by femurs infused with particles in these IL-1r1 knockout mice was no different than contralateral control femurs not infused with particles; the absolute levels of MCP-1 production from these experimental femurs were also significantly lower (6-8 fold lower) than those of wild type mice. However, inflammation and bone loss occurred to similar degrees in both IL-1r1 knockout and wild type mice, which indicated that while lack of the IL-1 receptor limited MCP-1 production, it did not abolish or reduce the overall inflammatory response to particles, due perhaps to activation of alternative inflammatory pathways (Epstein et al., 2005b). A followup study demonstrated histological evidence of inflammation and

Biological Response of Osteoblasts and Osteoprogenitors to Orthopaedic Wear Debris 211

In vivo cell responses to orthopaedic wear debris can also be evaluated using the bone harvest chamber (BHC). A modified version of the BHC, the drug test chamber (DTC), also allows evaluation of cell responses to infused therapeutic agents and growth factors in vivo under simultaneous exposure to wear particles. Earlier studies on in vivo tissue responses have been conducted by direct surgical implantation of particle boluses (e.g., 60-70 mg of PMMA cement powder) into the medullary tibial canal of rabbits via a drilled hole in the proximal tibia (Goodman et al., 1988, 1991a, 1991b). Though these studies have helped to elucidate the response of tissues to wear debris particles, the introduction of the BHC in the early 1990s has greatly facilitated this research process (Goodman et al., 1994, 1995a, 1995b, 1995c, 1996a, 1996b). The BHC is a titanium device with an inner core containing a 1 x 1 x 5 mm3 pathway for tissue ingrowth and an outer cylindrical shell with threads for screwing the cylinder into the surrounding bone. The outer shell contains 1 x 1 mm2 openings on its two ends that are continuous with the pathway in the inner chamber. When implanted into bone, the BHC allows bone ingrowth into its inner core; this bone tissue specimen can be collected at multiple times by removing the inner core, without disrupting the outer shell that has integrated with surrounding bone. In addition, particle suspensions can be placed inside the inner pathway such that ingrown bone reacts to these particulates, allowing the tissue reaction to be studied by histology after retrieval of the tissue sample. The DTC is essentially the BHC setup with an additional Alzet mini-osmotic pump that allows biologics to be infused into the inner core at a regulated rate. This mimics the clinical scenario in which therapeutic drugs are delivered locally to an area of tissue ingrowth. The DTC contains a 10 L reservoir for holding the infused solution and is linked to the inner core for bone ingrowth. The Alzet pump, which contains around 250 L of solution, is implanted subcutaneously in the animal and infuses fluid at a rate of 0.25 L/hr to the DTC reservoir via tubing. From there, the fluid travels to the inner chamber where the tissue is ingrowing and exposed to particles. As fluid builds up in the inner chamber, it is drained via outlet tubing to the skin of the animal. The Alzet pump and its tubing can also be removed and replaced with minimal disturbance to the system. The size of the BHC/DTC permits this device to be used only for rabbits or larger animals. With appropriate markers to identify multipotent stem cells, osteoprogenitors, and osteoblasts, the in vivo behavior of these cells in response to wear particles can potentially be studied using the harvest chamber models. The BHC has been used in a series of studies to evaluate in vivo tissue responses to wear particles during administration of oral p38 MAP kinase inhibitors in rabbits (Goodman et al., 2007; Ma et al., 2009). p38 MAP kinase mediates various pathways in inflammation, apoptosis, and osteoclast differentiation. In one study, BHCs were implanted and allowed to osseointegrate into the proximal tibial metaphyses of rabbits for 6 weeks. Ingrown tissue was removed and replaced with UHMWPE particles (0.5 0.2 m) at a concentration of 7.5 x 109 particles in 5 L in 1% sodium hyaluronate carrier solution at one of the 3-week treatment time intervals, with or without oral administration of p38 MAPK inhibitor, with comparison to control BHCs filled with carrier solution only. Tissue ingrowth into the BHC chamber was collected at the end of 3 weeks, and assessed histologically for expression of alkaline phosphatase (osteoblasts) or vitronectin receptor (osteoclasts) (Goodman et al., 2007). Histology tissue sections were also histomorphometrically quantified, using NIH Imaging software, for total tissue area, total bone area, ratio of total bone area over total tissue area, and total area of alkaline phosphatase-positive stains, and counted for the number of vitronectin receptor-positive cells. The oral p38 MAPK inhibitor, expected to

endosteal erosion characterized by fibrosis, jagged cortical margins, increased porosity, and presence of a periprosthetic membrane in femoral canals of IL-1r1 knockout mice injected with a bolus of titanium particles (Bragg et al., 2008).

The particle injection method has also been employed to study migratory patterns of injected cells, which may theoretically be extended to luciferase- and GFP-labeled marrow stromal or multipotent stem cells, osteoprogenitors, and osteoblasts. A series of studies has tracked the systemic migration of luciferase-transfected reporter macrophages injected into the lateral tail vein of nude mice with previous injection or infusion of particles into the femur (Ren et al., 2008, 2010, 2011). In one study, Simplex P bone cement powder 1-100 m in diameter (Howmedica Osteonics, Allendale, NJ, USA) consisting of 15% PMMA, 75% methylmethacrylate styrene copolymer, and 10% barium sulfate, as a 10% wt suspension in PBS, was injected as a single bolus of 10 L into the femoral canal of nude mice, followed 7 days later by injection of luciferase- and GFP-transfected Raw264.7 macrophages (5 x 105 cells in 100 L HBSS) into their lateral tail vein (Ren et al., 2008). Bioluminescence imaging of mice revealed significantly higher bioluminescent signal in particle-infused femurs at days 6 and 8 post-macrophage injection (4.7 ± 1.6, 7.8 2.9 respectively, ratios of signal of particleinfused femur over contralateral control femur), compared to those of saline-injected controls (1.2 0.2, 1.4 0.5). Histological analysis of femoral cross sections also showed higher numbers of GFP- and MOMA-2-positive macrophages (MOMA-2 is a macrophage marker) in the particle-infused femurs than in control femurs. The imaging and histology results indicated that macrophages from systemic sites migrated to tissues injured by wear debris particles. Similarly, UHMWPE particles (1.0 0.1 m) injected as a single bolus of 1.2 x 108 particles in 10 L into femora of nude mice attract luciferase- and GFP-transfected Raw264.7 macrophages to its site of infusion, resulting in significantly larger bioluminescent signals (10.32 ± 7.61 signal ratio) than saline-injected control femurs (signal ratio close to 1) 8 days after macrophage injection. Histological analysis revealed larger number of Raw264.7 macrophages positive for GFP and V3-positive osteoclasts in particle-infused femurs compared to saline-injected control femurs (Ren et al., 2010). UHMWPE particles continuously infused into femora of nude mice by the Alzet mini-osmotic pump (rather than by single bolus injection), also attracted labeled Raw264.7 macrophages to their site of infusion (Ren et al., 2011). Ten days after macrophage injection, femurs extracted for histology demonstrated increased numbers of GFP-labeled Raw264.7 macrophages, total macrophages (MOMA-2-positive), and vitronectin receptor/TRAP-positive osteoclasts in particle-infused samples than in saline-treated control femurs. Some cells stained positive for both TRAP and MOMA-2, and represent monocytes that have differentiated into osteoclasts. Bioluminescence imaging revealed significantly higher signal ratios in particleinfused femurs (13.95 ± 5.65) compared to saline-treated femurs with signal ratios close to 1. MicroCT scans of femurs infused with particles revealed decreased bone mineral density compared to saline-infused femurs (Ren et al., 2011). Taken together, these results indicate that systemic macrophages migrate to sites of particle infusion in response to particleinduced inflammation. The same model can potentially be applied to study the migratory patterns of systemically infused marrow stromal or multipotent stem cells, osteoprogenitors, and osteoblasts, which may respond to secreted cues of bone injury or osteolysis. The applicability of this model for this purpose rests upon identifying markers that allow appropriate isolation of pure osteogenic cell and progenitor populations for labeling with luciferase or fluorescent proteins.

endosteal erosion characterized by fibrosis, jagged cortical margins, increased porosity, and presence of a periprosthetic membrane in femoral canals of IL-1r1 knockout mice injected

The particle injection method has also been employed to study migratory patterns of injected cells, which may theoretically be extended to luciferase- and GFP-labeled marrow stromal or multipotent stem cells, osteoprogenitors, and osteoblasts. A series of studies has tracked the systemic migration of luciferase-transfected reporter macrophages injected into the lateral tail vein of nude mice with previous injection or infusion of particles into the femur (Ren et al., 2008, 2010, 2011). In one study, Simplex P bone cement powder 1-100 m in diameter (Howmedica Osteonics, Allendale, NJ, USA) consisting of 15% PMMA, 75% methylmethacrylate styrene copolymer, and 10% barium sulfate, as a 10% wt suspension in PBS, was injected as a single bolus of 10 L into the femoral canal of nude mice, followed 7 days later by injection of luciferase- and GFP-transfected Raw264.7 macrophages (5 x 105 cells in 100 L HBSS) into their lateral tail vein (Ren et al., 2008). Bioluminescence imaging of mice revealed significantly higher bioluminescent signal in particle-infused femurs at days 6 and 8 post-macrophage injection (4.7 ± 1.6, 7.8 2.9 respectively, ratios of signal of particleinfused femur over contralateral control femur), compared to those of saline-injected controls (1.2 0.2, 1.4 0.5). Histological analysis of femoral cross sections also showed higher numbers of GFP- and MOMA-2-positive macrophages (MOMA-2 is a macrophage marker) in the particle-infused femurs than in control femurs. The imaging and histology results indicated that macrophages from systemic sites migrated to tissues injured by wear debris particles. Similarly, UHMWPE particles (1.0 0.1 m) injected as a single bolus of 1.2 x 108 particles in 10 L into femora of nude mice attract luciferase- and GFP-transfected Raw264.7 macrophages to its site of infusion, resulting in significantly larger bioluminescent signals (10.32 ± 7.61 signal ratio) than saline-injected control femurs (signal ratio close to 1) 8 days after macrophage injection. Histological analysis revealed larger number of Raw264.7 macrophages positive for GFP and V3-positive osteoclasts in particle-infused femurs compared to saline-injected control femurs (Ren et al., 2010). UHMWPE particles continuously infused into femora of nude mice by the Alzet mini-osmotic pump (rather than by single bolus injection), also attracted labeled Raw264.7 macrophages to their site of infusion (Ren et al., 2011). Ten days after macrophage injection, femurs extracted for histology demonstrated increased numbers of GFP-labeled Raw264.7 macrophages, total macrophages (MOMA-2-positive), and vitronectin receptor/TRAP-positive osteoclasts in particle-infused samples than in saline-treated control femurs. Some cells stained positive for both TRAP and MOMA-2, and represent monocytes that have differentiated into osteoclasts. Bioluminescence imaging revealed significantly higher signal ratios in particleinfused femurs (13.95 ± 5.65) compared to saline-treated femurs with signal ratios close to 1. MicroCT scans of femurs infused with particles revealed decreased bone mineral density compared to saline-infused femurs (Ren et al., 2011). Taken together, these results indicate that systemic macrophages migrate to sites of particle infusion in response to particleinduced inflammation. The same model can potentially be applied to study the migratory patterns of systemically infused marrow stromal or multipotent stem cells, osteoprogenitors, and osteoblasts, which may respond to secreted cues of bone injury or osteolysis. The applicability of this model for this purpose rests upon identifying markers that allow appropriate isolation of pure osteogenic cell and progenitor populations for labeling with

with a bolus of titanium particles (Bragg et al., 2008).

luciferase or fluorescent proteins.

In vivo cell responses to orthopaedic wear debris can also be evaluated using the bone harvest chamber (BHC). A modified version of the BHC, the drug test chamber (DTC), also allows evaluation of cell responses to infused therapeutic agents and growth factors in vivo under simultaneous exposure to wear particles. Earlier studies on in vivo tissue responses have been conducted by direct surgical implantation of particle boluses (e.g., 60-70 mg of PMMA cement powder) into the medullary tibial canal of rabbits via a drilled hole in the proximal tibia (Goodman et al., 1988, 1991a, 1991b). Though these studies have helped to elucidate the response of tissues to wear debris particles, the introduction of the BHC in the early 1990s has greatly facilitated this research process (Goodman et al., 1994, 1995a, 1995b, 1995c, 1996a, 1996b). The BHC is a titanium device with an inner core containing a 1 x 1 x 5 mm3 pathway for tissue ingrowth and an outer cylindrical shell with threads for screwing the cylinder into the surrounding bone. The outer shell contains 1 x 1 mm2 openings on its two ends that are continuous with the pathway in the inner chamber. When implanted into bone, the BHC allows bone ingrowth into its inner core; this bone tissue specimen can be collected at multiple times by removing the inner core, without disrupting the outer shell that has integrated with surrounding bone. In addition, particle suspensions can be placed inside the inner pathway such that ingrown bone reacts to these particulates, allowing the tissue reaction to be studied by histology after retrieval of the tissue sample. The DTC is essentially the BHC setup with an additional Alzet mini-osmotic pump that allows biologics to be infused into the inner core at a regulated rate. This mimics the clinical scenario in which therapeutic drugs are delivered locally to an area of tissue ingrowth. The DTC contains a 10 L reservoir for holding the infused solution and is linked to the inner core for bone ingrowth. The Alzet pump, which contains around 250 L of solution, is implanted subcutaneously in the animal and infuses fluid at a rate of 0.25 L/hr to the DTC reservoir via tubing. From there, the fluid travels to the inner chamber where the tissue is ingrowing and exposed to particles. As fluid builds up in the inner chamber, it is drained via outlet tubing to the skin of the animal. The Alzet pump and its tubing can also be removed and replaced with minimal disturbance to the system. The size of the BHC/DTC permits this device to be used only for rabbits or larger animals. With appropriate markers to identify multipotent stem cells, osteoprogenitors, and osteoblasts, the in vivo behavior of these cells in response to wear particles can potentially be studied using the harvest chamber models. The BHC has been used in a series of studies to evaluate in vivo tissue responses to wear particles during administration of oral p38 MAP kinase inhibitors in rabbits (Goodman et al., 2007; Ma et al., 2009). p38 MAP kinase mediates various pathways in inflammation,

apoptosis, and osteoclast differentiation. In one study, BHCs were implanted and allowed to osseointegrate into the proximal tibial metaphyses of rabbits for 6 weeks. Ingrown tissue was removed and replaced with UHMWPE particles (0.5 0.2 m) at a concentration of 7.5 x 109 particles in 5 L in 1% sodium hyaluronate carrier solution at one of the 3-week treatment time intervals, with or without oral administration of p38 MAPK inhibitor, with comparison to control BHCs filled with carrier solution only. Tissue ingrowth into the BHC chamber was collected at the end of 3 weeks, and assessed histologically for expression of alkaline phosphatase (osteoblasts) or vitronectin receptor (osteoclasts) (Goodman et al., 2007). Histology tissue sections were also histomorphometrically quantified, using NIH Imaging software, for total tissue area, total bone area, ratio of total bone area over total tissue area, and total area of alkaline phosphatase-positive stains, and counted for the number of vitronectin receptor-positive cells. The oral p38 MAPK inhibitor, expected to

Biological Response of Osteoblasts and Osteoprogenitors to Orthopaedic Wear Debris 213

a growth factor that modulates osteoblast proliferation, differentiation, bone formation, and angiogenesis, in the presence of UHMWPE particles in rabbits (Goodman et al., 2003a). FGF-2 was infused at a dose of 50 ng/day over 3 weeks into the DTC, in the presence of a low (5.8 x 1011 particles/mL) or high (1.7 x 1012 particles/mL) concentration of UHMWPE particles (0.5 0.2 m). After 3 weeks, tissues extracted for histology and histomorphometric analysis revealed that FGF-2 significantly increased bone growth and decreased the number of vitronectin receptor-positive osteoclasts in samples treated with UHWMPE particles, compared to particle-treated samples not infused with FGF-2 (Goodman et al., 2003a). In another study, the DTC was infused with IL-10, an anti-inflammatory cytokine that suppresses Th1 helper cell-mediated inflammation, including expression of IL-1, IL-6, IL-8, TNF-, and GM-CSF, in the presence of UHMWPE particles in rabbits (Goodman et al., 2003b). IL-10 was infused at increasing doses of 0.1, 1.0, 10.0 and 100.0 ng/mL for 3 weeks at each dose, in the presence or absence of UHMWPE particles (1.7 x 1012 particles/mL) in the DTC. Histology and histomorphometirc analysis revealed that IL-10 infused at 1.0 ng/mL for 3 weeks, significantly increased bone growth up to 48% in the presence of UHMWPE particles, compared to particle-treated samples without IL-10. In the absence of particles, IL-10 had no effect on bone growth relative to controls not treated with IL-10 (Goodman et al., 2003b). In another study, the DTC was infused with OP-1 (also called BMP-7), a growth factor that promotes osteoblast proliferation, differentiation, and mineralization, in the presence of UHMWPE particles in rabbits (Ma et al., 2006). Infusion of OP-1 (110 ng/day) into the DTC for 6 weeks increased bone growth by 38% in the presence of UHMWPE

particles, relative to particle-treated samples without OP-1 (Ma et al., 2006).

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inhibit inflammation and bone loss, actually yielded dimnished bone ingrowth and alkaline phosphatase staining, and failed to suppress inflammation or foreign body reactions in the presence of UHMWPE particless (Goodman et al., 2007). A later study testing the effects of the oral p38 MAPK inhibitor SCIO-323, has shown similar results of reduced bone growth with no curtailment of inflammation in particle-treated groups, compared to particle-treated controls not receiving SCIO-323 (Ma et al., 2009). In summary, the BHC experiments have shown that p38 MAPK inhibitors do not improve bone formation in tissues exposed to wear debris particles.

Fig. 4. The drug test chamber (DTC). This system consists of a titanium chamber that permits tissue ingrowth, and an Alzet mini-osmotic pump for infusing biologics into the chamber. The titanium chamber consists of an outer cylinder that allows the device to be screwed into surrounding bone, and an inner core composed of a 10 L reservoir for holding infused solution and an inner canal/pathway for tissue ingrowth. The inner core is separable from the outer cylinder and allows the ingrown tissue to be collected without disrupting the entire device. The Alzet pump infuses solutions into the reservoir of the inner core, which in turn is connected to the tissue ingrowth canal via an open pore. Fluid outflow is drained via tubing to the subcutaneous tissue.

The DTC has been employed in a series of studies to evaluate in vivo tissue responses to wear particles during local infusion of trophic, osteogenic, or anti-inflammatory factors (Goodman et al., 2003a, 2003b; Ma et al., 2006). In one study, DTC was used to infuse FGF-2, a growth factor that modulates osteoblast proliferation, differentiation, bone formation, and angiogenesis, in the presence of UHMWPE particles in rabbits (Goodman et al., 2003a). FGF-2 was infused at a dose of 50 ng/day over 3 weeks into the DTC, in the presence of a low (5.8 x 1011 particles/mL) or high (1.7 x 1012 particles/mL) concentration of UHMWPE particles (0.5 0.2 m). After 3 weeks, tissues extracted for histology and histomorphometric analysis revealed that FGF-2 significantly increased bone growth and decreased the number of vitronectin receptor-positive osteoclasts in samples treated with UHWMPE particles, compared to particle-treated samples not infused with FGF-2 (Goodman et al., 2003a). In another study, the DTC was infused with IL-10, an anti-inflammatory cytokine that suppresses Th1 helper cell-mediated inflammation, including expression of IL-1, IL-6, IL-8, TNF-, and GM-CSF, in the presence of UHMWPE particles in rabbits (Goodman et al., 2003b). IL-10 was infused at increasing doses of 0.1, 1.0, 10.0 and 100.0 ng/mL for 3 weeks at each dose, in the presence or absence of UHMWPE particles (1.7 x 1012 particles/mL) in the DTC. Histology and histomorphometirc analysis revealed that IL-10 infused at 1.0 ng/mL for 3 weeks, significantly increased bone growth up to 48% in the presence of UHMWPE particles, compared to particle-treated samples without IL-10. In the absence of particles, IL-10 had no effect on bone growth relative to controls not treated with IL-10 (Goodman et al., 2003b). In another study, the DTC was infused with OP-1 (also called BMP-7), a growth factor that promotes osteoblast proliferation, differentiation, and mineralization, in the presence of UHMWPE particles in rabbits (Ma et al., 2006). Infusion of OP-1 (110 ng/day) into the DTC for 6 weeks increased bone growth by 38% in the presence of UHMWPE particles, relative to particle-treated samples without OP-1 (Ma et al., 2006).

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inhibit inflammation and bone loss, actually yielded dimnished bone ingrowth and alkaline phosphatase staining, and failed to suppress inflammation or foreign body reactions in the presence of UHMWPE particless (Goodman et al., 2007). A later study testing the effects of the oral p38 MAPK inhibitor SCIO-323, has shown similar results of reduced bone growth with no curtailment of inflammation in particle-treated groups, compared to particle-treated controls not receiving SCIO-323 (Ma et al., 2009). In summary, the BHC experiments have shown that p38 MAPK inhibitors do not improve bone formation in tissues exposed to wear

Fig. 4. The drug test chamber (DTC). This system consists of a titanium chamber that permits tissue ingrowth, and an Alzet mini-osmotic pump for infusing biologics into the chamber. The titanium chamber consists of an outer cylinder that allows the device to be screwed into surrounding bone, and an inner core composed of a 10 L reservoir for holding infused solution and an inner canal/pathway for tissue ingrowth. The inner core is separable from the outer cylinder and allows the ingrown tissue to be collected without disrupting the entire device. The Alzet pump infuses solutions into the reservoir of the inner core, which in turn is connected to the tissue ingrowth canal via an open pore.

The DTC has been employed in a series of studies to evaluate in vivo tissue responses to wear particles during local infusion of trophic, osteogenic, or anti-inflammatory factors (Goodman et al., 2003a, 2003b; Ma et al., 2006). In one study, DTC was used to infuse FGF-2,

Fluid outflow is drained via tubing to the subcutaneous tissue.

debris particles.

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**9**

*USA*

**Osteogenesis Imperfecta**

Roy Morello1,2 and Paul W. Esposito3,4 *1Department of Physiology & Biophysics,*

*University of Nebraska Medical Center, The Nebraska Medical Center, Omaha, NE, 4Department of Orthopaedic Surgery,*

*University of Arkansas for Medical Sciences, Little Rock, AR,*

*3Department of Orthopaedic Surgery and Rehabilitation,*

*Childrens Hospital and Medical Center, Omaha, NE,*

*2Division of Genetics, University of Arkansas for Medical Sciences, Little Rock, AR,*

Osteogenesis imperfecta (OI) is a congenital, generalized connective tissue disorder characterized by severe osteoporosis and bone fragility. Other features of the disease include dentinogenesis imperfecta, scoliosis, short stature, blue sclerae, hearing loss, and skin and ligament laxity (Cheung and Glorieux, 2008; Rauch and Glorieux, 2004). It is the most commonly inherited connective tissue disorder with a prevalence in the United States of about 25,000-50,000 cases (Martin and Shapiro, 2007). Molecular defects in type I collagen genes, *COL1A1* and *COL1A2*, were first associated with OI in the early 1980's (Chu et al., 1983). Since then, although OI is most commonly an autosomal dominant disease, it became evident that in certain pedigrees with recurrence of OI in children of unaffected parents, other mechanisms of inheritance were at play. While germline mosaicism for a type I collagen mutation was demonstrated in some rare cases (Cohn et al., 1990; Edwards et al., 1992), in others where no collagen primary sequence defects were evident, the existence of recessively inherited forms of OI had to be postulated. It is known that 10-15% of all OI cases are not caused by type I collagen mutations and some of these can be distinguished from others based on specific bone histological features (Glorieux et al., 2002), clinical features (Glorieux et al., 2000), or because they are linked to a genetic region not containing type I collagen genes (Labuda et al., 2002). It took about a quarter of a century to identify the first gene responsible for some cases of recessive OI, i.e. *CRTAP* (encoding Cartilage-associated protein) (Morello et al., 2006). This initial discovery led the way to the identification of several novel genes whose mutations cause recessive forms of OI. It has rapidly become evident that while dominant forms of OI are caused by mutations in type I collagen genes, recessive forms of OI are almost always caused by mutations in genes encoding for proteins involved in the type I collagen synthetic pathway and often residing in the rough endoplasmic reticulum (rER). Thus, all direct and indirect collagen interactors, either in the rER or Golgi apparatus or involved in intracellular collagen

**1. Introduction**

kinase signalling pathways are required for osteoblast-specific gene expression and differentiation in MC3T3-E1 cells. *J Bone Miner Res*, Vol. 17, No. 1, pp. 101- 110.

### **Osteogenesis Imperfecta**

Roy Morello1,2 and Paul W. Esposito3,4

*1Department of Physiology & Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, 2Division of Genetics, University of Arkansas for Medical Sciences, Little Rock, AR, 3Department of Orthopaedic Surgery and Rehabilitation, University of Nebraska Medical Center, The Nebraska Medical Center, Omaha, NE, 4Department of Orthopaedic Surgery, Childrens Hospital and Medical Center, Omaha, NE, USA*

#### **1. Introduction**

222 Osteogenesis

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kinase signalling pathways are required for osteoblast-specific gene expression and differentiation in MC3T3-E1 cells. *J Bone Miner Res*, Vol. 17, No. 1, pp. 101-

is an esssential mediator of orthopedic particle-induced osteoclastogenesis. *J Orthop*

kinase mediates PMMA induction of osteoclasts. *J Orthop Res*, Vol. 24, No. 7, pp.

osteoblast function by titanium particles. *J Bone Joint Surg Am,* Vol. 79, No. 1, pp.

osteoblasts to polymethylmethacrylate in vitro. Calcif Tissue Int, Vol. 62, No. 4,

Polymethylmethacrylate particles stimulate bone resorption of mature osteoclasts

Mouse femoral intramedullary injection model: technique and microCT scan

Prosthetic particles modify the expression of bone-related proteins by human

Osteogenesis imperfecta (OI) is a congenital, generalized connective tissue disorder characterized by severe osteoporosis and bone fragility. Other features of the disease include dentinogenesis imperfecta, scoliosis, short stature, blue sclerae, hearing loss, and skin and ligament laxity (Cheung and Glorieux, 2008; Rauch and Glorieux, 2004). It is the most commonly inherited connective tissue disorder with a prevalence in the United States of about 25,000-50,000 cases (Martin and Shapiro, 2007). Molecular defects in type I collagen genes, *COL1A1* and *COL1A2*, were first associated with OI in the early 1980's (Chu et al., 1983). Since then, although OI is most commonly an autosomal dominant disease, it became evident that in certain pedigrees with recurrence of OI in children of unaffected parents, other mechanisms of inheritance were at play. While germline mosaicism for a type I collagen mutation was demonstrated in some rare cases (Cohn et al., 1990; Edwards et al., 1992), in others where no collagen primary sequence defects were evident, the existence of recessively inherited forms of OI had to be postulated. It is known that 10-15% of all OI cases are not caused by type I collagen mutations and some of these can be distinguished from others based on specific bone histological features (Glorieux et al., 2002), clinical features (Glorieux et al., 2000), or because they are linked to a genetic region not containing type I collagen genes (Labuda et al., 2002). It took about a quarter of a century to identify the first gene responsible for some cases of recessive OI, i.e. *CRTAP* (encoding Cartilage-associated protein) (Morello et al., 2006). This initial discovery led the way to the identification of several novel genes whose mutations cause recessive forms of OI. It has rapidly become evident that while dominant forms of OI are caused by mutations in type I collagen genes, recessive forms of OI are almost always caused by mutations in genes encoding for proteins involved in the type I collagen synthetic pathway and often residing in the rough endoplasmic reticulum (rER).  Genetic classification of OI (AD = autosomal dominant; AR = autosomal recessive;

**Type I:** These individuals typically do not have major bone deformity and have a significant variability in terms of number of fractures, even within the same family. Fractures typically begin when the children start to ambulate. Plain radiographs often are normal in appearance. These individuals may have blue sclera and hearing loss, although this may not occur before the second or third decade. Dentinogenesis imperfecta is not typically a major component of Type I. There may be subtle dental findings. There are no long-term studies to determine if the collagen defect might lead to later dental problems. Growth and stature are

**Type II:** Type II is historically described as perinatally lethal, typically from pulmonary failure related to the severe involvement of the chest cavity with small volume, deformity, and recurrent rib fractures. There may also be some involvement of the intrinsic collagen of

These children are frequently diagnosed in prenatal ultrasounds with short, severely bowed long bones with multiple fractures. Radiographically, at the time of birth, they have severe

*Unknown* Unknown

*SERPINF1* PEDF

*FKBP10* FKBP10

*PLOD2* LH2

*LRP5 (null alleles)* LRP5

rhyzomelia *CRTAP* CRTAP

Hypertrophic callus, dense metaphyseal band, calcification of radio-ulnar interosseus membrane

Severe: mineralization defect, 'fish scale' bone lamellae, slightly elevated AP

Moderate to severe with joint contractures (Bruck syndrome 1?)

Moderate to severe with joint contractures and pterygia, Bruck syndrome 2

Ocular form of OI, osteoporosis pseudoglioma syndrome

typically mildly decreased in type I OI (Forlino et al., 2011; Sillence et al., 1979).

the lung parenchyma (Forlino et al., 2011; Sillence et al., 1979).

VII AR Very severe to lethal,

trafficking, have become new candidates for recessive or atypical forms of OI. This chapter will provide the reader with an update on the latest developments in the OI field, including description of new genes causing OI, mechanisms of disease and novel therapeutic approaches.

#### **2. Classification of OI, clinical features and differential diagnosis**

Osteogenesis imperfecta was classified several years ago into four types based on clinical, radiological and genetic features (Sillence, 1988; Sillence et al., 1979). Type II OI is the perinatal lethal form followed by types III, IV and I, in decreasing order of severity with type I OI being the mildest form. The mode of inheritance is not unique to each type with at least types II (further sub-classified into IIA, IIB and IIC) and III that could be inherited in a dominant or recessive fashion. More recently, out of the more heterogeneous group of type IV patients, novel types V, VI and VII OI were described based on unique clinical and/or histological features and linkage to loci where no type I collagen genes were mapped (Glorieux et al., 2000; Glorieux et al., 2002; Labuda et al., 2002).

The nosology of OI has been a subject of discussion and revision in light of recent discoveries of novel genes causing recessive forms of the disease. Plotkin (Plotkin, 2004) proposed to classify as OI all those cases in which a mutation in either *COL1A1* or *COL1A2* can be identified. Cases resulting from mutations in other genes would be classified as syndromes resembling OI. Others have proposed to adopt a revised Sillence classification of OI type I through VI but removing types VII and VIII which are due to mutations in *CRTAP* and *LEPRE1*, respectively, but have clinical and radiological features indistinguishable from types II-IV (Van Dijk et al., 2010). A more genetically oriented classification has also being proposed (Forlino et al., 2011). Here the classical Sillence OI types I-IV are maintained (all due to either *COL1A1* or *COL1A2* defects), followed by the previously described types V, VI and VII, and then in increasing order new types of OI (the latest is number XI) where each new type (from types V-XI) is due to a mutation in a different gene (**Table 1**). Conversely, the 2010 Revision of the Nosology and Classification of Genetic Skeletal Disorders (Warman et al., 2011) has agreed to retain the Sillence classification as the universally accepted way to classify the degree of OI severity but to free it from any direct molecular reference.

Because the original Sillence classification has been in use for over 30 years and instantly refers to the clinical severity of the disease, it is likely that physicians will continue to use it at least until the underlying genetic mutation of a patient has been clearly determined. The strictly genetic classification could be quite useful though as it is indeed possible that different genetic etiology and hence mechanism of disease may require a different therapeutic approach. This is beginning to emerge for instance in patients with *SERPINF1* mutations who have not shown beneficial effects from bisphosphonate treatment (Homan et al., 2011).

Although there remains a great deal of difficulty making a definitive diagnosis in many of the children, recognition that there are recessive forms of osteogenesis imperfecta has clarified a historically confusing differential diagnosis. Utilization of these modified classifications also assists in identifying individuals without the classic types I – IV from normal children with nonaccidental trauma and other unrelated metabolic bone diseases.

trafficking, have become new candidates for recessive or atypical forms of OI. This chapter will provide the reader with an update on the latest developments in the OI field, including description of new genes causing OI, mechanisms of disease and novel

Osteogenesis imperfecta was classified several years ago into four types based on clinical, radiological and genetic features (Sillence, 1988; Sillence et al., 1979). Type II OI is the perinatal lethal form followed by types III, IV and I, in decreasing order of severity with type I OI being the mildest form. The mode of inheritance is not unique to each type with at least types II (further sub-classified into IIA, IIB and IIC) and III that could be inherited in a dominant or recessive fashion. More recently, out of the more heterogeneous group of type IV patients, novel types V, VI and VII OI were described based on unique clinical and/or histological features and linkage to loci where no type I collagen genes were mapped

The nosology of OI has been a subject of discussion and revision in light of recent discoveries of novel genes causing recessive forms of the disease. Plotkin (Plotkin, 2004) proposed to classify as OI all those cases in which a mutation in either *COL1A1* or *COL1A2* can be identified. Cases resulting from mutations in other genes would be classified as syndromes resembling OI. Others have proposed to adopt a revised Sillence classification of OI type I through VI but removing types VII and VIII which are due to mutations in *CRTAP* and *LEPRE1*, respectively, but have clinical and radiological features indistinguishable from types II-IV (Van Dijk et al., 2010). A more genetically oriented classification has also being proposed (Forlino et al., 2011). Here the classical Sillence OI types I-IV are maintained (all due to either *COL1A1* or *COL1A2* defects), followed by the previously described types V, VI and VII, and then in increasing order new types of OI (the latest is number XI) where each new type (from types V-XI) is due to a mutation in a different gene (**Table 1**). Conversely, the 2010 Revision of the Nosology and Classification of Genetic Skeletal Disorders (Warman et al., 2011) has agreed to retain the Sillence classification as the universally accepted way to classify the degree of OI severity but to

Because the original Sillence classification has been in use for over 30 years and instantly refers to the clinical severity of the disease, it is likely that physicians will continue to use it at least until the underlying genetic mutation of a patient has been clearly determined. The strictly genetic classification could be quite useful though as it is indeed possible that different genetic etiology and hence mechanism of disease may require a different therapeutic approach. This is beginning to emerge for instance in patients with *SERPINF1* mutations who have not shown beneficial effects from bisphosphonate treatment (Homan et

Although there remains a great deal of difficulty making a definitive diagnosis in many of the children, recognition that there are recessive forms of osteogenesis imperfecta has clarified a historically confusing differential diagnosis. Utilization of these modified classifications also assists in identifying individuals without the classic types I – IV from normal children with nonaccidental trauma and other unrelated metabolic bone diseases.

**2. Classification of OI, clinical features and differential diagnosis**

(Glorieux et al., 2000; Glorieux et al., 2002; Labuda et al., 2002).

free it from any direct molecular reference.

al., 2011).

therapeutic approaches.

Table 1. Genetic classification of OI (AD = autosomal dominant; AR = autosomal recessive; NC = not classified)

**Type I:** These individuals typically do not have major bone deformity and have a significant variability in terms of number of fractures, even within the same family. Fractures typically begin when the children start to ambulate. Plain radiographs often are normal in appearance. These individuals may have blue sclera and hearing loss, although this may not occur before the second or third decade. Dentinogenesis imperfecta is not typically a major component of Type I. There may be subtle dental findings. There are no long-term studies to determine if the collagen defect might lead to later dental problems. Growth and stature are typically mildly decreased in type I OI (Forlino et al., 2011; Sillence et al., 1979).

**Type II:** Type II is historically described as perinatally lethal, typically from pulmonary failure related to the severe involvement of the chest cavity with small volume, deformity, and recurrent rib fractures. There may also be some involvement of the intrinsic collagen of the lung parenchyma (Forlino et al., 2011; Sillence et al., 1979).

These children are frequently diagnosed in prenatal ultrasounds with short, severely bowed long bones with multiple fractures. Radiographically, at the time of birth, they have severe

Osteogenesis Imperfecta 227

Centers that treat large numbers of these children however have anecdotally noted a marked increase in activity level and ambulation, even in children with severe type III OI. Present indications for treatment with bisphosphonates in children with the more severe forms of OI include marked bowing, which is interfering with comfort and function, and recurrent fractures (de Graaff et al., 2011; Forin et al., 2005; Glorieux, 2007; Glorieux et al.,

Fig. 1. A, B: 15-month old with type III OI. Note the relatively normal physis, and reasonable bone development following treatment with pamidronate since infancy. However, the significant bowing will not remodel, and as these children begin to pull up to stand they will predictably fracture at the apex of the bow. C: untreated 17 year old also with type III

The greatest improvement in areal bone density occurs in the first year of treatment. Pamidronate has been studied the most extensively, and has been shown to improve vertebral height and development in children with compressed vertebral bodies (Land et al., 2006b). Infants and young children with more severe OI with bowing and recurrent fractures have been safely treated with pamidronate (Astrom et al., 2007; Land et al., 2006a; Plotkin et al., 2006). The DEXA scan is the most frequent test utilized to help determine the relative severity of the OI, and the Z-score has ben utilized to monitor the response to treatment. However, there is not data suggesting that the goal of treatment is to develop a bone density in the normal range. At the present time, the clinical parameters of improved comfort, decreased fracture rate, and progression of normal developmental milestones offers the best clinical assessment of effective treatment. Zoledronic acid is a more potent bisphosphonate, which requires only one intra-venous treatment per year. It is presently being studied, but this data and results are not yet available (Shapiro and

Initial treatment protocols with pamidronate were for a set number of years depending on the treating center. However, it has been noted that if the treatment is discontinued in a growing child, the bone that is developing at the proximal and distal end of the long bones is not treated. This gives rise to a stress riser between the treated bone of the shaft and the relatively soft bone at either end of the long bones (Breslau-Siderius et al., 1998; Rauch et al.,

Theoretically, this is a concern with the longer acting agents, which may be given only

demonstrating popcorn epiphysis and signs of recurrent fractures.

A B C

Sponsellor, 2009).

2006)**.**

1998; Plotkin et al., 2006; Rauch and Glorieux, 2006).

bowing of the long bones, multiple fractures in different stages of healing, poorly defined cortices, and crumpling or wrinkling of the bones in an accordion-type fashion. There is poor tubulation with wide medullary canals. There is typically severe wedging deformity of the vertebrae. The skull is usually quite soft and large (Forlino et al., 2011; Sillence et al., 1979). Some of these children, including some treated with bisphosphonates, have survived for several years.

**Type III:** These patients are also frequently diagnosed on prenatal ultrasound. They also have relative shortening of the long bones, significant bowing, and multiple prenatal and postnatal fractures. These children continue to have fractures even with gentle handling. This is the most severe non-lethal form. They may have a very typical triangular facies, blue sclerae, dentinogenesis imperfecta, vertebral fractures, and frequently scoliosis. Without treatment with bisphosphonates, this OI type frequently demonstrates classic "popcorn epiphyses" of the long bones and patients have significant short stature (Forlino et al., 2011; Sillence et al., 1979).

**Type IV:** Traditionally, this classification has included children with phenotypes that did not fit into Sillence type I or III. These children typically have multiple fractures, varying degrees of deformity, as well as a great deal of variation in terms of color of the sclera, cranial settling, dentinogenesis imperfecta, and stature. With the recognition of recessive forms, many of these children previously classified as having type IV are now recognized as having one of these other, less common forms (Forlino et al., 2011; Sillence et al., 1979).