Osteoactivin is Critical for Postnatal Bone Growth and

Development Prospectus 2013

Hilary Stinnett

PI: Dr. Fayez Safadi

11/6/2013

This study will test the hypothesis that OA is important to bone differentiation and function, and that the OA derived pep-tide OA-D is capable of inducing bone growth in C57/b6, OAKO, and D/2J mice. A combined approach analyzing mice in vivo and cells in vitro will be used to describe the function of OA via the OAKO mouse strain. This study will also examine the therapeutic potential of OA-D for bone loss diseases by administration in estrogen deficient osteoporotic mice.

H. Stinnett Prospectus 2013 Page 1

Specific Aims

Osteoporosis is a disease marked by bone loss caused by increased trabecular spacing and large, honey-comb like structures,

leading to brittle bones and increased fracture risk [1, 2]. In osteoporosis and other bone loss diseases, the balance between osteoblast

deposition and osteoclast resorption is altered, resulting in net bone loss. Osteoporosis impacts 55% of the population aged 50 and

older, with post-menopausal, thin women of Caucasian descent at the highest risk for developing this disease[3, 4]. A variety of factors

can contribute to its development; low estrogen (in women) or testosterone levels (in men); thyroid conditions (heightened thyroid or

parathyroid levels); sedentary lifestyle, and diet low in calcium and vitamin D are major culprits [4]. Most osteoporotic therapies focus

on inhibiting osteoclast function, as is the case for the bisphosphonates; PTH (1-34), known as teriparatide (Forteo), is the only bone

anabolic factor currently available on the market [4]. Additional bone anabolic factors are needed to combat osteoporosis and other

bone loss diseases.

One protein with the potential to fulfill this need is osteoactivin (OA), also known as GPNMB or DC-HIL. Our laboratory

has previously identified this protein in bone; it is also expressed in heart, brain, thymus, and skeletal muscle [5] . OA is a

transmembrane type I protein that plays important roles in osteoblast proliferation and differentiation, adhesion, and both

endochondral and intramembranous ossification [6-8]. It is expressed in both osteoblasts and osteoclasts, and plays a vital role in bone

growth and maintenance [6-8]. It has two known isoforms: a transmembrane, glycosylated form (115kDa), and a cleaved, secreted

portion (68kDa). OA has a number of domains, including a polycystic kidney disease domain (PKD), proline-rich repeat domain

(PRRD), a di-leucine motif, an N-terminal signal peptide, and the C-terminal RGD motif[9]. Our laboratory has previously derived a

number of peptides from OA encompassing different domains; osteoactivin-D (OA-D) is one promising result, an 18 amino acid

biologically active peptide, which encompasses the RGD domain.

The purpose of this study is to elucidate the role of OA in bone metabolism and evaluate the therapeutic potential of bone

anabolic factor OA-D. On the basis of this data, we hypothesize that:

Hypotheses: OA is critical for postnatal bone growth and development. Peptide OA-D is capable of stimulating bone formation and

rescuing osteoporotic phenotypes. This will be studied through three specific aims: (I) Characterization of the skeletal phenotype of the

OA Knockout (OAKO) mouse strain using in vivo and

in vitro approaches. (II) Testing the efficacy of OA-D peptide on bone

mass in vivo in C57/b6, OAKO, and D/2J mice strains

(III) Examining the efficacy of OA-D peptide on bone

mass in estrogen-deficient osteoporosis in C57/b6, OA Rescue,

OAKO, DBA and D/2J mice.

Utilizing OAKO mice will reveal novel information about how OA

functions in bone formation. This in turn will enable us to develop novel

therapeutic strategies for the treatment of fracture and bone loss diseases.

OA-D is one promising option which will be examined for its ability to

increase bone mass with potential for clinical application.

Background Bones are continuously undergoing remodeling through an active process

that engages a multitude of factors. These cycles of events can be simply

described by the Activation-Resorption-Formation (ARF) cycle:

osteocyte death triggers osteoclastogenesis via RANK-Ligand (RANKL)

(Activation phase); the resulting production of cytokines, such as TGF-β

and OPG, regulate osteoclast number, leading to bone resorption

(Resorption phase); osteoclasts secrete factors to recruit bone-lining cells

and osteoblasts to the area, resulting in fresh matrix formation and mineralization (Formation Phase) (See Table 1) [10].

Osteoblasts are bone-forming cells that differentiate from mesenchymal stem cells (MSC) and undergo three distinct stages:

proliferation (day 1-7), extracellular matrix deposition and matrix maturation (days 7-14), and mineralization (days 14-21)[11]. In

mature bone, terminally differentiated osteoblasts preferentially form bone in pits where collagen has been removed, laying down a

matrix of Collagen Type I (ColI) and hydroxyapatite crystals [12]. During the process of bone formation, osteoblasts occasionally

become entrapped in the mineralized matrix, becoming osteocytes [11, 12].

Osteocytes are important mechanosensory cells responsible for the stimulation of bone resorption or formation[12]. These

cells communicate via dendritic processes that travel through mineralized bone in small channels called cannuliculi [11].

Communication between osteoblasts and osteocytes appears to be paracrine in nature [13]. Osteocyte apoptosis recruits osteoclasts to

stimulate bone resorption, beginning the remodeling process [14].

Both osteocytes and osteoblasts recruit osteoclasts to a site of bone formation via M-CSF and RANKL. Osteoclasts, the bone

Factor Name Abbv: Identifies:

Osteocalcin OC Presence of Terminally

Differentiated Osteoblasts

Alkaline

Phosphatase

ALP Bone Formation

Bone

Sialoprotein

BSP Bone Mineralization

Osteopontin OPN Bone Resorption

Osteoprotegrin OPG Osteoclast Inhibition

Osterix OSX Osteoblast activity

RANK-Ligand RANKL Osteoclast stimulation

Carboxy-

Terminal

Collagen

Crosslinks

CTX Bone Resorption

Runt-related

transcription

factor

RUNX2 Osteoblast differentiation

marker

Pu.1 Pu.1 Osteocyte differentiation/

commitment

Capthesin K CapK Osteoclast activity

Table 1. Notable Identification Factors

H. Stinnett Prospectus 2013 Page 2

resorption cells, differentiate from hematopoietic stem cells (HSC) [12]. A close link between osteoclast and osteoblast number has

been observed, suggesting that differentiation of osteoclasts appears to be inhibited by the signaling factors of nearby osteoblasts [11].

Research Strategy: Significance & Rational

Current pharmaceutical therapies for osteoporosis involve amino bisphosphonates, such as aldronenate (Fosamax®), which

prevent further bone loss from resorption, and bone anabolic peptide teriparatide (PTH 1-34). However, amino bisphosphonates

merely halt disease progression and are incapable

of reversing disease damage on their own; some

studies also suggest that this class of medication

can cause osteoblast apoptosis [15]. PTH (1-34) is

an enormous cost-burden on the patient, and

concerns have been raised about chronic use

inducing bone loss [16]. New bone anabolic

proteins are needed to increase the number and

availability of therapeutic options for people with

osteoporosis.

OA is one possibility due to its role in

bone maintenance. However, more information is

necessary to understand how OA functions. A

study utilizing the OAKO mice would further

describe the role OA plays in both osteoblasts and

osteoclasts, as well as clarify the potential for OA

peptide as a bone growth therapy. Due to its role

in osteoblast function, increasing OA levels in vivo

may induce bone growth. The size of OA, however, may be both too large to reach its intended target in the body and cost inhibitory.

Osteoactivin-D (OA-D) is an 18 amino acid peptide derived from the C-terminal end of OA; its small size may enable it to reach the

intended target with intraperitoneal administration. Preliminary studies have revealed that injection of the OA-D peptide induces an

increase in BV/TV (Fig 3). This study will further examine the therapeutic potential for OA-D to increase bone mass in D/2J, OAKO,

and estrogen-deficient osteoporosis.

Previous studies have shown that in the missense-mutation mouse strain D/2J, trabecular mass and number are significantly

decreased in vivo; introducing the fully-functional OA-D peptide may rescue this bone phenotype. This would provide further

evidence of the efficacy of the OA peptide.

Development of new bone anabolic factors also has implications in both osteoporotic therapy and bone fracture. Further

examination of OA’s role in osteoblast and osteoclast function via OAKO, as well as OA-D’s capacity to rescue D/2J and

ovariectomized models may reveal OA-D as a novel therapeutic agent.

Mice Strains

Previous studies have shown that OA plays an important role in osteoblast and osteoclast function. The primary goal of the

proposed study is to examine the function of OA via a knockout mouse model, and evaluate the therapeutic potential of the bone

anabolic peptide OA-D for osteoporosis. Several strains of mice will be utilized to

accomplish this goal.

One mouse strain, generated by Jackson Laboratory (Bar Harbor, ME,

USA), D2J, has a missense mutation in OA, resulting in a truncated and

nonfunctional form of the protein. In these mice, bone volume/ tissue volume

(BV/TV), trabecular number, and trabecular thickness are significantly decreased

relative to normal (DBA) mice at 4 weeks, 8 weeks, and 16 weeks of age[14] (See

Fig 1). D/2J mice display increased osteoblast number but decreased function,

suggesting that the absence of functional OA is inhibiting osteoblast function [14].

C57black/ 6 (C57/b6), also ordered from Jackson laboratories, will be utilized as a

control mouse for various experiments. The OAKO mouse strain is derived from

C57/b6 and shares its genetic background. Our laboratory developed the OAKO

mouse strain which will be used in this experiment; this mouse strain will enable us

to identify the importance of OA on both in vivo and in vitro. OA-Rescue (OA-R)

mice were derived by crossing D2J mice with DBA/2J-Dtnbp1sdy

/J (with the OA

wildtype allele). Progeny were backcrossed for a minimum of 6 generations to

produce OA-R.

H. Stinnett Prospectus 2013 Page 3

Innovation:

Describing OA-dependent bone remodeling processes. Previous data has shown that OA plays important roles in bone remodeling,

however more information is needed to further clarify its function. The OAKO mouse model, derived by our laboratory, will give

unique insight into the age-dependent relationship between OA and bone remodeling in vivo, as well as demonstrating any deficiencies

on the cellular level via in vitro studies examining osteoblast and osteoclast differentiation and function (See Fig 2).

Novel Bone Anabolic Protein OA-D as a potential therapy for Osteoporosis. OA has great potential as a therapy for bone loss diseases;

one derivative, OA-D, will be examined to determine its efficacy in inducing bone growth. This peptide will be tested to determine

whether it can induce bone growth in C57/b6, OAKO, and D/2J mice, as well as estrogen-deficient osteoporosis model, induced by

ovariectomy, in C57/black 6 (C57/b6), OA- Rescue, OA KO, DBA, and D/2J mice.

Research Strategy: Approach

Aim 1: Characterization of skeletal phenotype in OAKO mice using in vivo and in vitro approaches. On the basis of preliminary data, we hypothesize that OAKO mice will have decreased bone mass in vivo.

I. µCT at age 4 weeks, 8 weeks, 12 weeks, and 16 weeks. Microcomputer tomography (µCT) will be performed on OAKO

femurs and calveria with age-matched controls in C57/b6 at 7.7um using SkyScan Control Software. Both male and

female mice will be assayed. Trabecular meshwork modeled by µCT will be examined for variation in number,

thickness, spacing, and bone volume over tissue volume. II. Kinetic histomorphometry. Calcein will be injected intraperitoneally 7 days prior and 2 days prior to sacrifice in order to

determine rate of bone remodeling at ages 4 weeks, 8 weeks,12 week, and 16 week old mice. This will detect bone

formation and mineral apposition rates. III. MSC and Osteoblast Cultures. Primary osteoblasts will be gathered from 4-6 day old mouse calveria from OAKO and

C57/B6 mice. MSCs will be harvested from femur bone marrow of 6-8 week old mice; HSC and MSC will be separated

in culture. MSCs will be differentiated into osteoblasts from bone-marrow derived cells with β-glycerolphosphate

(10mM), dexamethasone(10-6

M), and ascorbic acid (50µg/mL). CyQuant proliferation and Alamar Blue stains will be

performed on cultures at 24, 48, and 72 hours for all described cell types. ALP and ALP activity assays will be

performed on osteoblast cultures at day 14. Alizarin Red and von Kossa mineralization assays will be performed on

osteoblast cultures at day 21. IV. HSC and Osteoclast Cultures. HSCs will be harvested from femur bone marrow of 6-8 week old mice; HSC and MSC

will be separated in culture. HSCs in culture will be differentiated to osteoclasts with M-CSF(20ng/mL) and RANKL

(20ng/mL). CyQuant proliferation and Alamar Blue stains will be performed on cultures at 24, 48, and 72 hours for all

described cell types. TRAP Activity and TRAP staining will be performed at day 7. V. Qualitative Real Time Polymerase Chain Reaction. QPCR will be performed on long bone (tibia) and flat bone

(calveria) from all age groups and sexes to analyze differences in bone formation markers. Cell cultures will also be

examined according to their appropriate markers. Osteoblast culture and bone RNA will be examined fro: OC, ALP,

RUNX2, OSX; osteoclasts and bone RNA will be examined for TRAP, Calcitonin R, Capthesin K, and Pu1 (see Table

1). VI. Enzyme-Linked Immunosorbent Assay (ELISA). Bone

formation, differentiation, and resorption markers OPG, OA,

RANKL, CTX and OC will be examined for the cell lysates

from the cultures described above, as well as serum from the 4

week, 8 week, 12 week, and 16 week old mice. VII. Histological Analysis. Plastic sectioning will be performed on

calcein-labeled femurs to identify any changes in growth plate

between OAKO and C57/b6.

Aim II: In vivo use of osteoactivin-D peptide. We hypothesize that in vivo injection of the OA-D peptide will induce an

increase in bone mass in C57/b6, OAKO, and D/2J mice.

I. Injections of PBS, PTH, and OA-D, Age 8 Weeks. C57/B6

mice will be injected with OA-D peptide (5mg/kg body

weight, stock of 1µg/µL). Recombinant parathyroid hormone (1-34, 40mg/kg body weight, stock 8ng/µL) will be used as a

positive control and Phosphate Buffered Saline (5mL/kg body weight, stock 10mM) will be a negative control (Fig 3). Each treatment will be injected intraperitoneally, five consecutive days per week, for 4 weeks total. Two days were

given between final injection and sacrifice. Weights will be recorded prior to every injection.

Figure 3. Changes in Bone Mass in PBS, PTH, OA-D Injected Mice.

H. Stinnett Prospectus 2013 Page 4

II. Histology of Liver, Kidney, Spleen, Reproductive organs. Paraffin sections will be taken from liver (7µm), kidney (7µm),

spleen (7µm), and reproductive organs (5µm) of all injected mice and stained with hematoxylin and eosin (H&E) to

check for any toxicity that may result from injection of the OA-D peptide. III. µCT for Injected Mice. µCT will be performed on femurs from

the injected mice at 7.7 µm to examine changes in trabecular number, thickness, spacing, and differences in bone

volume/tissue volume (See Fig 2). IV. ELISA. Bone formation and resorption markers OPG, RANKL, OC, and OA will be run on serum from the injected

mice.

Aim III: Phenotypic Rescue of OA mutant and Osteoporotic mouse models.

We hypothesize that in vivo injection of the OA-D peptide will induce an increase in bone mass in ovariectomized C57/b6, OAKO,

OA-R, D/2J, and DBA mice. I. D/2J Mouse Model Rescue. OA-D and PBS will be injected in D/2J and DBA (control) mice as described in Aim II at 9

weeks of age for 4 weeks. Experimental outcomes will be analyzed following the methods described in Aim II. II. OAKO Mouse Model Rescue. OAKO mice will be injected with OA-D and PBS as described in Aim II at 9 weeks of

age for 4 weeks; WTLM and C57/black 6 will also be injected as controls. Experimental outcomes will be analyzed

following the methods described in Aim II. III. Ovariectomized Mouse Model Rescue. C57/B6, D/2J, DBA, OAKO, and OA-Rescue mice will be ovariectomized at 12

weeks to induce estrogen-deficient osteoporosis. These mice will be injected in the same method described above

starting 4 weeks after surgery, lasting 4 weeks. Control mice from each of the strains will be ovariectomized at 12

weeks and sacrificed at 8 weeks post-surgery to ensure development of the osteoporotic phenotype. Experimental

outcomes will be analyzed following the methods described in Aim II. IV. Histological Analysis. Paraffin sections will be taken from liver (7µm), kidney (7µm), reproductive organs (5µm) and

spleen (7µm) and stained with H&E to investigate any possible toxicity. Plastic sectioning of femurs will be stained

with H&E and Trichrome to identify changes in the femur between the injected and non-injected mice. Experimental

outcomes will be analyzed following the methods described in Aim II.

Key Methods

Primary osteoblasts: Primary osteoblasts will be isolated form neonatal mouse pups at day 4-6 as described previously [7].

Bone marrow cultures: HSCs and MSCs will be gathered from 6-8 week old mouse femurs and separated in vitro. These cells will be

examined and differentiated into osteoclasts and osteoblasts, respectively, per standard protocol[7].

RNA Isolation: RNA will be isolated from cell culture as described previously [7]. RNA from tibia and calveria will be taken by

crushing the bone in liquid nitrogen with mortar and pestle into a fine powder, then completing the RNA isolation protocol with Trizol

[7]. RNA will be converted to cDNA, then run for QPCR.

RT-PCR and Q-PCR: RT-PCR will be completed as described previously [7]. The primers for determining genotype for OA-KO were

sense: GCA GCC TCT GTT CCA CAT ACA CTT CA and anti-sense: GCC CAC CAC ATG CTT TAT AAG TGT CC; the primers for

OA-WT were modeled by Jackson Laboratories and were sense: TCC CAT CTC GAA GGT GAA AG and antisense: AAA TGG CAG

AGT CGT TGA GG. Each mouse (toe clip samples) will be tested for both primers; presence of the OAKO primer alone indicated KO,

presence of OAKO and OAWT indicated heterozygosity, and presence of only OAWT indicated wildtype genotype. QPCR will be

performed on Applied Biosystems Step One Plus using SYBR green method and quantified using the –∆∆ CT method as described by

the Applied Biosystems QPCR manual [17]. Osteoblast and osteoclast markers ALP, OPG, TRAP9, ColI, Osx, BSP, OPN and Oc will

be examined.

Hematoxylin and Eosin Staining: Paraffin sections will stained as described in Histological and Histochemical Analysis [18].

Alkaline Phosphatase (ALP) activity measurement and histochemistry: ALP will be measured as described previously [19]. Osteoblast

cultures will be stained as described previously and observed in the Olympus Bh2-RFCA microscope [20].

Von Kossa staining of mineralized nodules: Primary osteoblast cultures will be stained for mineralization according to previous

protocol [20].

Generation and administration of peptides: The OA-D peptide generated previously was utilized [9]. Anaspec in Fremont, CA

synthesized the peptide; the sequence is as follows: (H-Lys-Ala-Pro-Phe-Ser-Arg-Gly-Asp-Arg-Glu-Lys-Asp-Pro-Leu-Leu-Gln-Asp-

Lys-OH). OA-D peptide was diluted to 1µg/µL in sterile PBS. Human PTH (1-34) was purchased from Fisher Scientific

H. Stinnett Prospectus 2013 Page 5

(Cat #AP22-1-25-A). The PTH was diluted to 8ng/mL in sterile water. 10mM sterile PBS was used. All peptides were administered

intraperitoneally at 5µL/g body weight using a 1mL syringe and 25 gauge needle.

Plastic Sectioning: The Osteo-Bed Bone Embedding kit from Polysciences will be used to perform a modified protocol developed by

our laboratory. Briefly, the femurs will be processed in the NEOMED tissue processor Shandon Citadel for 18 hours. The processing

schedule is as follows: 90 minutes in 75% ethanol twice, 90 minutes in 95% ethanol twice, 90 minutes in 100% ethanol three times, 90

minutes 100% ethanol-xylene, followed by 90 minutes Clear Advantage (xylene substitute) twice. The samples in cassettes will then

undergo the following procedure under vacuum: MMA resin overnight; the following day, the MMA will be changed twice with a 2.5

hour incubation time; placed in catalyzed resin (1.4g benzoyl peroxide in 100mL MMA resin) for 2 hours; embedding resin (3.5g

benzoyl peroxide/100mL MMA resin) overnight. The next day, the femurs will be placed in glass scintillation vials (Wheaton Science

Products, Cat# 986541) with condyles down and covered with 10mL embedding resin. These vials will be placed under vacuum for

30min-1hour, then the caps tightened. The vials will be placed in a sand bath in an oven at approximately 34ºC and left for 3 days to

polymerize. Post-polymerization, the samples will be removed from the glass vials (by breaking and removing the glass), then cut into

small squares by Buehler Isomet 1000. The squares will be mounted on chucks with superglue (PolySciences Cat#15899-50). The

blocks will be soaked face-down in 20% ethanol and cut at 5-7µm using a Leica RM2165 microtome. The samples are placed into the

water bath 45˚C, then placed directly onto the slides. A piece of polyethylene plastic will be placed over the slide and rolled firmly

with rubber roller to ensure adhesion and remove wrinkles and bubbles. The slides are then stacked with wax paper between each slide

in a slide press and placed in a 60˚C oven overnight. In the morning, the wax paper and polyethylene pieces are removed.

Microcomputer Tomography: Each sample will be carefully dissected from the mouse and cleaned without scraping to ensure the

integrity of the bone. Bones are saved in glass scintillation vials in 70% ethanol at 4˚C until use. Each bone is wrapped in gauze and

placed vertically into a plastic tube. 75% of the bone is scanned using a Microphotonics Skyscan microcomputer tomography system

at 7.7µm with filter. Each scan is reconstructed, then analyzed by hand using CTAn, the Skyscan analysis software, at 0.4mm below

the growth plate. Length of analyzed section will be age dependent: 3mm for 4 week, 5.25mm for 8 week, and 5.75mm for 12 and 16

week.

Ovariectomy: Mouse Ovariectomy will be performed in the surgical suite at the CMU. The mice will undergo surgery as described

previously [21].

Masson Trichrome Staining: After deplastisizing, trichrome staining on plastic sectioning will be performed as described on IHC

world protocol [22].

Histomorphometry: Trichrome stained plastic sections will be analyzed by BIOQUANT 98 (Bioquant Image Analysis Corporation,

Nashville, TN) for number of osteoblasts and osteoclasts as well as nodules as previously described [20]

Summary

This study will test the hypothesis that OA is important to bone differentiation and function, and that the OA derived peptide OA-D is

capable of inducing bone growth in C57/b6, OAKO, and D/2J mice. A combined approach analyzing mice in vivo and cells in vitro

will be used to describe the function of OA via the OAKO mouse strain. This study will also examine the therapeutic potential of

OA-D for bone loss diseases by administration in estrogen deficient osteoporotic mice.

Expected Results

OAKO Phenotypic Study:

It is possible that the OAKO mice will require challenge in order to display a phenotypic difference (Fig1). It is anticipated

that the OAKO mice will display decreased osteoblast and osteoclast differentiation and function as shown in proliferation

and survival assays. These differences are also expected to appear in QPCR data in the form of decreased bone formation

markers and increased osteoclast markers. Calcein staining is expected to reveal decreased bone turnover in the OAKO mice

compared to the C57/b6 mice, and growth plate thickness may be decreased in the OAKO mice as displayed by plastic

sectioning of the femur. OA-D Peptide Injection Study:

Preliminary µCT data from the C57/b6 OA-D injected mice are trending towards significance; it is possible this study has not

yet reached significance owing to the small sample size. Histological sectioning from the study, in combination with the

steadily increased body weight, suggests no toxicity from the OA-D peptide. We anticipate that ELISA will reveal an increase

in osteoblast function and possibly decrease in osteoclast function, resulting in the observed bone mass increases (Fig2). OA-

D peptide is expected to increase bone mass in D/2J and OAKO to comparable levels to the C57/b6 control. Phenotypic Rescue Study:

OA-D injection of the ovariectomized mouse models is anticipated to significantly prevent bone loss; the OA-D treated

ovariectomized mice are expected to be comparable to the non-ovariectomized mice of corresponding genotype. The

differences are expected to be more pronounced in D/2J and OAKO mice. No toxicity observable by histological analysis is

H. Stinnett Prospectus 2013 Page 6

anticipated on the basis of preliminary data.

Alternative approaches

If differences between OAKO and C57/b6 femurs are undetectable, the spine will be scanned by µCT to next to identify

changes. It is possible that the OAKO mice will require challenge in order to display a phenotypic difference due to alternative

compensatory mechanisms. Ovariectomy is one option for challenging the phenotype to stimulate differences, as described in Aim III.

The OA-D injected C57/b6 mice demonstrated an increase in bone mass relative to PBS, but that increase was not

statistically significant; it is possible that an increase in sample size (to n=6 optimal) would be insufficient to reach significance. In

this case, a C57/b6 non-injected mouse of similar age (12 weeks) could be utilized as an additional control to identify any changes.

The OA-D peptide needs optimization for dosage, administration technique, and frequency.

OA-D injection in OAKO mice may have no observable effects on bone mass in vivo; in this case, osteoblast and osteoclast

activity will be examined in vitro. Microarray analysis may also reveal differences and enable identification of alternative mechanisms

for bone growth to investigate.

Several potential problems may arise in regard to injection of the OA-D peptide: it is possible that the peptide may interact

with unintended receptors or fail to reach its intended target in bone. These problems may be addressed by alternating the section of

OA utilized in the peptide by choosing another OA-derived peptide.

In the case of the ovariectomized mice, non-ovariectomized (surgical sham) mice may be utilized as control mice for the

ovariectomized mouse rescue experiment (normal mouse vs. osteoporotic vs. osteoporotic with treatment). In this case, one-way

ANOVA will be used to test for significance.

Statistical Analyses

For all studies, preliminary data was analyzed by Mike Hewit, NEOMED biostatistician, to determine minimum sample size. All tests

will be run in Graphpad PRISM, a statistics and graphing software.

OAKO Characterization Study: Student t-test will be utilized to test µCT, ELISA, ALP Activity, and calcein data for significance. Dunnett one-way ANOVA

will be used to determine significance of ∆-CT method analysis between QPCR markers. A minimum sample size n=6 will be

used for each sex and age group.

OA-D Injection Study:

One-way Tukey ANOVA will be utilized to identify significant changes in µCT and ELISA tests. Dunnett one-way ANOVA

will be used to determine significance on ∆-CT method analysis of QPCR markers. A minimum sample size of n=3 will be

used for each treatment type.

Ovariectomized Model: Student t-test will be utilized to test µCT and ELISA results. Dunnett one-way ANOVA will be used to determine significance

on ∆-CT method analysis of QPCR markers. A minimum sample size of n=3 will be used for each phenotype and treatment.

H. Stinnett Prospectus 2013 Page 7

Vertebrate Animals

The Comparative Medicine Unit (CMU) at the Northeast Ohio Medical University (NEOMED) is a 24,000- square-foot, multi-species

animal care and use facility that has been continuously accredited by the Association for Assessment and Accreditation of Laboratory

Animal Care, International (AAALAC) since 1982. The CMU was again awarded Continued FULL accreditation status by AAALAC,

International on July 1, 2011. The CMU is registered as a research facility with the United States Department of Agriculture (USDA

Registration #31-R-0092) and has an Animal Welfare Assurance number (#A3474-01) on file with the Public Health Service (PHS).

The facility is staffed by eight (8) qualified personnel and is under the direction of a veterinarian who is certified as a specialist in

laboratory animal medicine by the American College of Laboratory Animal Medicine (ACLAM).

1) Detailed description - The proposed studies require the use of newborn, growing (various ages), and adult mice. Pathogen-free

colonies of OA-null (OAKO) mice, OA-mutant (D2J), control (DBA), rescue (OA-R), and wild-type (C57/Blk6) mice have been

established and maintained at NEOMED. Both the PI and the co-I have experience in maintaining knockout colonies. All mice

will be toe-clipped for identification and genotyping at 9 days, and weaned 3 weeks after birth. We estimate that the total number

of mice required to complete the studies as proposed will be 560, including mice for the generation of bone marrow derived oste-

oclast cultures and breeders needed to maintain the colonies. The majority of the mice used for the proposed experiments in aim 1

will be at age 4 weeks, 8 weeks, 12 weeks, and 16 weeks of age, both male and female. Aim 3 will involve ovariectomy to induce

osteoporotic phenotype in 12 week old mice; the technique was taught by a veterinarian and mice will be monitored post-surgery

by the staff- veterinarian. All animal care and use will strictly follow the guidelines established by NIH and Temple University’s

IACUC.

2) Justify the use of animals – The mice will be used as experimental models to study the effects of OA knockout on skeletal phe-

notype in vivo, as well as investigation into osteoclast and primary osteoblast cultures in vitro. The OAKO mice are unique mod-

els in a loss-of-function approach that investigates the importance of OA in skeletal phenotype and bone maintenance. Collective-

ly, these types of studies are expected to have direct clinical applications with respect to the development of therapeutic strategies

to stimulate bone growth in patients with osteoporosis or in fracture repair. In preliminary studies, we determined that 6 ani-

mals/group were required for statistical significance (p<0.01) with 85% power to demonstrate a 5-10% difference in histological

measures between OAKO and C57/b6 mice. We anticipate the loss of up 3 samples in each group throughout the study, thus we

plan to use at least 9 animals in each group. Breeding for maintenance of OAKO is outlined below:

a. Breeding 1: Heterozygous/heterozygous OAKO mice may be bred to create knockout mice. Knockout/heterozygous will also be utilized

as breeders. Knockout/Knockout have few, small litters and will not be used for this study. Wildtype litter mates will be eu-

thanized along with heterozygous mice not used for breeding.

b. Breeding 2: C57/b6, OA-R, D/2J, and DBA colonies have already been established as mouse lines and will continue to be bred for use in

this study.

3) Veterinary care - All animal care and use, as well as the maintenance of the breeding colonies for mice are under the direct su-

pervision of NEOMED's veterinarian, Dr. Walter Horne. The animals are housed in a barrier facility that is AAALAC-accredited.

4) Procedures for ensuring that discomfort and pain are limited - Most experiments involve the removal of cells and tissues

from euthanized animals without any prior experimentation. Some studies require regular in vivo injection with PBS, OA-D, or

PTH peptide; these studies are classified as Category C (momentary, slight pain/distress). Some studies require in vivo injections

of calcein for labeling of new bone formation prior to being euthanized; these studies are classified as Category C (momentary,

slight pain/distress). Some mice will be ovariectomized, classified as Category D (survival surgery, pain treated); these mice will

be under anesthesia (isoflurane) during surgery and pain monitored daily until the animal is euthanized. Rimadyl will be adminis-

tered subcutaneously in response to pain behaviors up to 3 days post-surgery. All mice (breeders) will be monitored on a daily ba-

sis; general physical appearance, activity and eating habits will be noted and any animal that appears unhealthy will be euthanized.

5) Method of euthanasia - Animals will be euthanized by CO2 inhalation followed by cervical dislocation. Mice under 6 days old

will be euthanized by decapitation. These methods are consistent with the recommendations of the Panel on Euthanasia of the

American Veterinary Medical Association (AVMA).

H. Stinnett Prospectus 2013 Page 8

Citations

1. Suh, T.T. and K.W. Lyles, Osteoporosis considerations in the frail elderly. Curr Opin Rheumatol, 2003. 15(4): p. 481-6.

2. Mori, S., [Contribution of bone quality to fracture risk]. Clin Calcium, 2004. 14(10): p. 33-8.

3. National Osteoporosis Foundation. Available from: http://www.nof.org.

4. Staff, M.C. Definition. 2013 July 18, 2013]; Available from: http://www.mayoclinic.com/health/osteoporosis/DS00128.

5. Safadi, F.F., et al., Cloning and characterization of osteoactivin, a novel cDNA expressed in osteoblasts. J Cell Biochem,

2001. 84(1): p. 12-26.

6. Ornitz, D.M. and P.J. Marie, FGF signaling pathways in endochondral and intramembranous bone development and human

genetic disease. Genes Dev, 2002. 16(12): p. 1446-65.

7. Abdelmagid, S.M., et al., Osteoactivin, an anabolic factor that regulates osteoblast differentiation and function. Exp Cell Res,

2008. 314(13): p. 2334-51.

8. Sheng, M.H., et al., Targeted overexpression of osteoactivin in cells of osteoclastic lineage promotes osteoclastic resorption

and bone loss in mice. PLoS One, 2012. 7(4): p. e35280.

9. Selim, A.A., et al., The role of osteoactivin-derived peptides in osteoblast differentiation. Med Sci Monit, 2007. 13(12): p.

BR259-70.

10. Roodman, G.D., Cell biology of the osteoclast. Exp Hematol, 1999. 27(8): p. 1229-41.

11. The Molecular Biology of the Cell. 5 ed. 2008, Garland Science, Taylor & Francis Groupl, LLC.

12. Henriksen, K., et al., Local communication on and within bone controls bone remodeling. Bone, 2009. 44(6): p. 1026-33.

13. Tran Van, P.T., A. Vignery, and R. Baron, Cellular kinetics of the bone remodeling sequence in the rat. Anat Rec, 1982.

202(4): p. 445-51.

14. Abdelmagid, S.M., et al., Mutation in OA/GPNMB Reduces Bone Formation in vivo and Osteoblast Differentiation in vitro.

15. Idris, A.I., et al., Aminobisphosphonates cause osteoblast apoptosis and inhibit bone nodule formation in vitro. Calcif Tissue

Int, 2008. 82(3): p. 191-201.

16. Lane, N.E.a.A.K., A Review of Anabolic Therapies for Osteoporosis. Arthritis Res Ther, 2003. 5(5): p. 214-222.

17. Biosystems, A. Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR. 2008. 70.

18. Kiernan, J., Histological and Histochemical Methods: Theory and Practice, 2008, Scion: Bloxham, UK.

19. Selim, A.A., et al., Anti-osteoactivin antibody inhibits osteoblast differentiation and function in vitro. Crit Rev Eukaryot Gene

Expr, 2003. 13(2-4): p. 265-75.

20. Abdelmagid, S.M., et al., Osteoactivin acts as downstream mediator of BMP-2 effects on osteoblast function. J Cell Physiol,

2007. 210(1): p. 26-37.

21. Bone Research Protocols, in Methods in Molecular Biology, H.a. Ralston, Editor 2012, Springer Science + Business Media. p.

545-551.

22. Masson's Trichrome Staining Protocol for Collagen Fibers. [cited 2013; Available from:

http://www.ihcworld.com/_protocols/special_stains/masson_trichrome.htm.