Nonclinical Assessment of Tumor Growth Enhancement Potential by B2A-Coated Ceramic Granules Used in Arthodesis

Abstract

B2A is a synthetic peptide that augments osteodifferentiation and improves bony fusion when delivered on ceramic granules during arthrodesis surgery. Nonclinical safety assessments, including genotoxicity, supported the use of B2A-coated granules as a combination medical device in pilot clinical studies. As a prelude to pivotal clinical studies, an assessment of the possibility that B2A-coated granules (and B2A) could enhance tumor growth was undertaken. B2A-coated granules demonstrated no evidence of genotoxicity. Cell culture studies with human tumor cell lines demonstrated that neither exposure of cells to B2A or B2A-coated granules increased cell proliferation or invasive capability relative to controls. In vivo, surgically implanted B2A-coated granules did not increase tumor growth (4 human tumor cell lines) or metastasis (1 cell line) relative to vehicle controls in immune-compromised rodents. Thus, traditional genotoxicity, as well as specially designed tumor growth enhancement studies, indicates that the possibility of tumor enhancement appears unlikely.

Keywords

tumor growth B2A human tumor xenograft coated granules

Introduction

B2A is a synthetic peptide that acts as a positive receptor modulator to augment osteodifferentiation, thereby promoting bone regeneration.^1,2 B2A is not a recombinant growth factor, hormone, or morphogenetic protein but acts on BMP receptors consistent with an allosteric activator. At the molecular and cellular level, B2A binds to cell surface receptors in the BMP receptor family and, in the presence of BMP-2, will augment the differentiation of preosteoblasts and mesenchymal stem cells toward bone tissue. In the absence of BMP-2, B2A does not induce osteodifferentiation.

B2A has been integrated into an investigational, combination medical device (B2A-coated ceramic granules) intended for use in arthrodesis surgery, an orthopedic procedure which fuses the bones that form a joint, essentially replacing the joint with new bridging bone. Arthrodesis is widely used by the orthopedic community in patients whose afflicted joints (in the spine, the wrist, and the foot/ankle) are so severely damaged that usual pain management techniques fail.

The calcium phosphate-based ceramic component of the B2A-coated granules provides a scaffold for cell migration and growth. B2A, that slowly elutes from the granules, is intended to augment the ongoing, naturally occurring differentiation of preosteoblasts in cooperation with endogenous BMP-2, typically upregulated during bone repair. Ultimately, the process results in an augmentation of bony fusion.

Nonclinical safety and effectiveness assessments, including experimental arthrodesis studies in animals,^3,4 supported the use of B2A-coated ceramic granules in pilot clinical studies. In those studies, B2A-coated ceramic granules were under clinical investigation in 2 formats (1) in lumbar spine arthrodesis studies⁵ as a bone graft extender mixed with local autograft bone (Prefix) and (2) in foot and ankle arthrodesis studies⁶ as a bone graft substitute (Amplex) with no need for autograft bone. In both formats, the type of ceramic granules and the type of peptide, for example B2A, are exactly the same. The key differences relate to whether or not local autograft bone is used and to a lower B2A dose in foot and ankle fusion procedures.

There were no device-related adverse events identified in the pilot studies. The clinical outcomes were supportive of continued investigation in larger, pivotal clinical trials.

Due to the regulatory concerns about a possible risk of malignancy, and in particular, the possibility of enhancement of cancer progression (tumor promotion), additional nonclinical studies were undertaken using B2A-coated ceramic granules as would be used in pivotal clinical trials of degenerative disc disease of the lumbar spine. The studies examined whether B2A-coated granules could accelerate tumor growth and phenotypes associated with metastasis. These studies included in vitro evaluations of human tumor cell lines and in vivo evaluations of tumor growth in xenograft models of human cancer. The evaluations involved the use of human cancers of lung, colon, and prostate origin—diverse human cancer types highly relevant to human disease. The results detected no tumor growth enhancement by B2A-coated granules or B2A.

Materials and Methods

Test Articles

B2A-coated granules were prepared from a 2-component kit immediately before use. The kit contains a vial of ceramic granules and a vial of lyophilized B2A.

The granules are composed of 20% hydroxyapatite and 80% β-tricalcium phosphate. The diameters of the granules are 1 to 2mm, and the granule pore diameters are approximately 400 µm. B2A was formulated as a lyophilized powder using mannitol and glycine as excipients. Just prior to use, B2A is hydrated with water for injection (USP) and coated by charge–charge adsorption onto the ceramic granules.

B2A acetate is a peptide synthesized by solid-phase peptide chemistry with an empirical formula of C₂₄₁H₄₁₈N₆₆O₆₅S₂ and a relative molecular mass of 5344.4 (free peptide). B2A contains 42 naturally occurring amino acids and 3 residues of 6-aminohexanoic acid (Ahx), an analog of lysine. All 45 amino acids in B2A are of nonanimal origin. The sequence of B2A is Ala-Ile-Ser-Met-Leu-Tyr-Leu-Asp-Glu-Asn-Glu-Lys-Val-Val-Leu-Lys-Lys(Ala-Ile-Ser-Met-Leu-Tyr-Leu-Asp-Glu-Asn-Glu-Lys-Val-Val-Leu-Lys)-Ahx-Ahx-Ahx-Arg-Lys-Arg-Leu-Asp-Arg-Ile-Ala-Arg-NH₂.

In the tumor promotion studies, B2A was coated on the granules at 750 µg/cm³. Consistent with the use in clinical spinal fusion procedures, the B2A-coated granules were mixed 1:1 with morselized bone (isograft).

Genotoxicity and Cytotoxicity Studies

B2A-coated ceramic granules (3 mg/cm³) were evaluated as a medical device^7,8 for genetic toxicity in a standard battery of studies (Toxikon Corporation, Bedford, Massachusetts). In vitro extracts of B2A-coated ceramic granules were tested for their potential to induce bacterial mutation in the Ames test (Salmonella typhimurium and Escherichia coli with and without an exogenous mammalian activation system, S9) and to induce chromosome aberrations in Chinese hamster ovary (CHO) cells. Appropriate positive controls were used in each test to verify the sensitivity of the test systems. For the Ames assay, the test article was extracted in 0.9% sodium chloride (NaCl) or cottonseed oil at a ratio of 0.2 g/mL at 70°C for 24 hours. For the CHO assay, the test article was extracted in Ham’s F-12 medium at a ratio of 0.2 g/mL at 70°C for 24 hours. In vivo extracts of B2A-coated ceramic granules were tested for induction of micronuclei in the bone marrow of Albino Swiss mice (6-12 weeks of age, specific pathogen free), which received a single intraperitoneal injection of vehicle or extract (0.9% NaCl at a ratio of 0.2 g/mL at 70°C for 24 hours) at 50 mL/kg.

An agar diffusion assay (Toxikon Corporation) was used to evaluate the cytotoxicity of B2A-coated ceramic granules (without extraction). In this method, a thin layer of nutrient-supplemented agar was placed over cultured L929 cells. The B2A-coated ceramic granules on filter paper were placed on top of the agar layer, and the cells were incubated. After 48 hours, the cultures were examined, and a zone of malformed, degenerative, or lyzed cells under and around the test material indicated cytotoxicity.

Follow-on cytotoxicity studies were also conducted with the B2A peptide. In the assay, 1000 WI-38 human fibroblast cells were seeded into individual wells in 96-well plates in growth medium containing B2A, and the cells were cultured for 4 days. At the end of 4 days, the cells were assayed for growth using the CyQUANT Cell Proliferation Assay Kit as per manufacturer’s instructions (Life Technologies, Grand Island, New York). The basis for the assay is a fluorescent dye that exhibits strong fluorescence enhancement when bound to cellular nucleic acids, allowing for direct fluorescence measurement. The amount of dye bound correlates directly with the cell number.

Cytotoxicity, if present, would represent a decrease in cell number relative to the control. Sodium azide (0.05%), a positive control compound, caused significant cytotoxicity.

In Vitro Cell Proliferation

B2A was evaluated for its ability to enhance in vitro cellular proliferation of serum-starved human tumor cell lines A549 (nonsmall-cell lung carcinoma), LoVo (colon adenocarcinoma), and PC-3 (prostate carcinoma). Cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, Virginia) and propagated in a humidified 5% carbon dioxide (CO₂) incubator at 37°C in the following growth media: Dulbecco Modified Eagle Medium (DMEM): F12 supplemented with 10% fetal bovine serum, 2 mmol/L l-glutamine, 1 mmol/L sodium pyruvate, and 50 µg/mL gentamicin. Typically, 3000 cells were plated in 96-well tissue culture plates in 100 µL growth media and allowed to adhere overnight. Following the overnight incubation, the growth media was replaced by serum-low medium (DMEM: F12 supplemented with 2% new born calf serum, 2 mmol/L l-glutamine, 1 mmol/L sodium pyruvate, and 50 µg/mL gentamicin). After conditioning in serum-low medium for 24 hours, B2A was added in serum-low medium and the cells were allowed to grow. To test the effects of B2A-coated granules, cells were seeded into the lower chamber of 24-well transwell plates (polycarbonate membrane, 0.4 µm pore size). After conditioning in serum-low medium for 24 hours, 0.1 cm³ of ceramic granules, sham-coated or coated with B2A (0.75 mg/cm³ granule), was added to the upper chamber in serum-low medium. Treatment with B2A was carried out for 4 days unless otherwise noted. Cell proliferation was analyzed using the CyQUANT Cell Proliferation Assay Kit as per manufacturer’s instructions.

Epithelial-Mesenchymal Transition (EMT) Assay

Human cancer cell lines PANC-1 (pancreatic carcinoma), Caco-2 (colorectal adenocarcinoma), LS174T (colorectal adenocarcinoma), LoVo (colorectal adenocarcinoma), and WiDr (colorectal adenocarcinoma) were obtained from ATCC. After seeding into 4-well chamber slides (177399, NUNC) in serum-low medium, the cells were treated with B2A for 3 days in serum-low medium. At the end of the treatment, cells were assayed for EMT markers by immunofluorescence as follows. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized in 0.5% Triton X-100 for 10 minutes, and incubated in protein-free blocking buffer (Pierce Biotechnology, Inc, Rockville, Illinois) at room temperature for 1 hour. The cells were incubated for 1 hour with anti-β-catenin (1:500; BD Biosciences, San Jose, CA), anti-E-cadherin (1:100; Santa Cruz Biotechnology Inc Santa Cruz, CA), or anti-fibronectin (1:400; Sigma-Aldrich Inc, St. Louis, MO). Thereafter, the cells were incubated for 1 hour with Alexa Fluor 488-conjugated goat antimouse and/or antirabbit secondary antibodies (1:1000; Molecular Probes, Life Technologies Corporation, Eugene, OR) The cells were then counterstained with bis-benzamide (8 ng/mL) for cell nuclei. The slides were viewed by fluorescence microscopy. The translocation of β-catenin from its usual membrane site at cell-to-cell junctions to the cytoplasm, as well as loss of E-cadherin, and the gain of the mesenchymal marker fibronectin was indicative of EMT.

Cell Invasion Assay

B2A was evaluated for its ability to enhance in vitro invasion by human tumor cell lines A549 (nonsmall-cell lung carcinoma), LoVo (colon adenocarcinoma), and PC-3 (prostate carcinoma).

Studies were carried out using a commercially available kit (QCM 24-well Collagen-Based Cell Invasion Assay, Millipore Corporation, Billerica, MA) following the manufacturer’s instructions. The assay is based on transwell invasion across a collagen-coated membrane. Trypsinized cells were resuspended in serum-low medium to a density of 1 × 10⁶/mL and 300 µL was added to each of the upper chambers; B2A or vehicle in serum-low medium used in the lower chamber. After 24 hours incubation at 37°C, cells on the upper surface of the filter were mechanically removed with a cotton swab and discarded. Cells that had invaded to the lower surface were stained, the stain dissolved, and the dissolved stain which correlated with (invading) cell number was quantified using a microplate reader at 560 nm.

Animal Studies

The animal studies were conducted under approved protocols. The animals were housed, maintained, and used in studies compliant with all policies of the “Guide for the Care and Use of Laboratory Animals,” DHHS (NIH 85-23), Animal Welfare (DHHS-TN 73-2) the NIH Manual Issuance 4206 and 6000-3-4-58, “Responsibility for Care and Use of Animals CDC/NIH 4th edition,” and Public Health Service Policy on Humane Care and Use of Laboratory Animals. The athymic mice were BALB/c nude (nu/nu) males aged 8 to 10 weeks and obtained from Taconic (Germantown, New York). Athymic young adult rats (NIHRNU, male) were obtained commercially from Taconic. Male severe combined immunodeficiency (SCID)/beige mice were obtained from Charles River Laboratories and used when aged approximately 2.5 months.

Two different animal models were used (1) athymic rats or mice subcutaneously injected with human tumor cells and (2) SCID/beige mice injected orthotopically with bioluminescent PC-3M-luc cells of human prostate carcinoma origin. In the athymic rat model, B2A was administered as an implant in a posterolateral fusion surgery. In the SCID/beige mouse model, B2A was administered as an implant subcutaneously juxtaposed against the spine. In both types of studies, B2A (as B2A-coated granules) was used at a dose of approximately 1.9 mg/kg, 30 times higher than the anticipated human dose (0.0625 mg/kg).

Athymic Rat Subcutaneous Model

The animal model involved inoculation of athymic rats on the flank with tumor cells (LoVo or A459), followed 2 days later by posterolateral fusion surgery and implant of B2A-coated ceramic granules or uncoated ceramic granules (control). Both the B2A-coated ceramic granules and the uncoated ceramic granules were mixed with isograft bone chips prior to implant. Tumor volume and weight were monitored for each animal.

The LoVo and A549 cell lines were obtained and propagated as described above. Cells were harvested, washed, and suspended in phosphate-buffered saline. For LoVo cells, aliquots of 200 µL (1.5 × 10⁷cells) were used for subcutaneous injection into the lower flank of the animals. For A549 cells, aliquots of 100 µL (0.5 × 10⁷ cells) were used for injection.

Surgical implant of test articles was performed 2 days after subcutaneous implantation of the tumor cells. To collect isograft bone chip, rats were euthanized by CO₂ overdose immediately before the bone harvesting. Bones (iliac crest and long bones) were dissected and morselized using ronjeurs into approximately 1 to 2-mm chunks. Before implant, the bone chips were mixed 1:1 (volume) with the B2A-coated ceramic granules. Uncoated granules mixed with an equal volume of bone chips were used as the control. The approximate B2A dose to the animal was 0.39 mg/animal (1.9 mg/kg, for a 200-g rat).

For surgery, anesthesia was induced in the rats with ketamine and xylazine (90 and 4 mg/kg intraperitoneal, respectively). The rats were positioned and prepared in standard surgical fashion. The L4-L5 posterolateral fusions were performed with the spine approached through a single midline skin incision using 2 paramedian fascial incisions. Once exposed, the transverse processes were decorticated with a scalpel until a blush of cancellous bone was observed. The wounds were irrigated and approximately 0.2 cm³ (measured by a 1 cm³ syringe) of graft material was placed into the fusion beds. The fascia was closed with 3.0 vicryl sutures, and the skin was closed with stainless steel surgical clips and the wounds dressed.

Tumors were measured twice weekly by a blinded observer using calipers, and tumor volume (mm) was calculated using the formula (L × W ²)/2, where L is the longest diameter and W is the shortest diameter.

At the end of the study (4 weeks for LoVo tumors and 6 weeks for A549 tumors), tumors were carefully excised and weighed in a blinded fashion to determine the accuracy of the estimated tumor volume to actual tumor weights. The difference in tumor volume between the 2 groups was analyzed by 1-way analysis of variance (ANOVA). Statistical significance was ascribed at P < .05.

The SCID/Beige Mouse Orthotopic Model

These studies involved the in vivo monitoring of tumor growth and metastasis using bioluminescent PC-3M-luc cells in murine models of human prostate cancer. The PC-3M-luc2 is a luciferase-expressing cell line that was stably transfected with firefly luciferase gene (luc2) and is derived from the human prostate adenocarcinoma cell line PC-3M selected from metastasis. The studies were conducted in collaboration with Caliper Life Sciences, Discovery Alliances and Services.

The experiment was conducted on male SCID/beige mice. All animals received orthotopic xenograft of PC-3M-luc2 cells (10⁶ cells per animal, direct surgical injection into prostate) prior to receiving subcutaneous implant of the test compound.

Two weeks after tumor cell inoculation, the animals were separated into 2 treatment groups: group A, 10 animals receiving sham surgery and group B, 10 animals receiving 0.2 cm³ of graft implant (0.1 cm³ B2A-coated granules + 0.1 cm³ bone). The syngeneic bone (femurs and tibia) was harvested from 10 littermates SCID/beige mice. In these studies, the implant was placed over the lumbar spine to accommodate the volume of implant.

In vivo bioluminescent imaging was performed on an IVIS Spectrum instrument. Images were acquired weekly with animals in ventral and dorsal positions. Prior to imaging, the animals received an injection of luciferin solution (100 µL, intraperitoneally, XenoLight Ultra solution). Then, 10 minutes later, animals were anesthetized (inhalation of 3% isoflurane) and imaged using the following parameters: auto exposure, open aperture, and medium binning.

Athymic Mouse Studies

The PC-3 prostate tumor cells were suspended in Growth Factor Reduced Matrigel and injected subcutaneously on the flanks of BALB/c nude (nu/nu) male mice. On day 6, the animals were randomly assigned to 2 groups, control animals received saline injections and B2A-treated animals received B2A (1 mg/kg, at day 6, 8, and 10) injections. There were 7 animals in each group. At day 28, the tumor masses were dissected and weighed.

Statistical Analysis

For cell proliferation assays, statistical significance was determined by paired Student t testing using the raw data and was compared to controls. The tumor volume and weight were analyzed by using 1-way ANOVA on ranks (Kruskal-Wallis).

Results

Genotoxicity/Cytotoxicity Studies

Assessment in standard International Organization for Standardization 10993 biocompatibility panels for genotoxicity and cytotoxicity indicated that B2A-coated granules were neither genotoxic nor cytotoxic (data not presented). The results are briefly described below.

The test articles, saline and CSO extracts of B2A-coated ceramic granules, were negative in the bacterial reverse mutation assay in 4 S typhimurium strains (TA98, TA100, TA1535, and TA1537) or in E coli strain WP2. There were no significant changes in the incidence of the CHO cells possessing abnormal chromosomes exposed to saline extracts of B2A-coated ceramic granules for 22-hour treatments with and without S9 metabolic activation. No micronucleus-inducing activity was observed in the in vivo mouse bone marrow micronucleus test following single intraperitoneal administration.

B2A-coated ceramic granules exhibited no cytotoxicity in agar diffusion assays at 48 hours post-exposure. Also, the B2A peptide exhibited no cytotoxicity in human WI-38 fibroblasts after 4 days (10 µg/mL, 1.9 µmol/L).

In Vitro Effects on Human Tumor Cells

Cell Proliferation

B2A did not substantially increase tumor cell proliferation in human lung, colon, or prostate tumor cells in vitro (Figure 1) at any concentration tested. B2A was used at a maximum of 10 µg/mL. This concentration corresponds to nearly 10 times the maximum theoretical human plasma concentration (1.1 µg/mL, based on a total B2A dose of 3.75 mg, 100% bioavailability, and no clearance). To more closely simulate the clinical situation, B2A-coated granules were also examined for their effects on tumor cell proliferation. B2A-coated granules did not increase proliferation of LoVo, A549, or PC-3 tumor cell lines.

Figure 1.

B2A had no discernable effect on in vitro cell proliferation of human tumor cell lines (LoVo, colon carcinoma; A549, non-small-cell lung carcinoma; PC-3, prostate carcinoma) when provided on granules (left panel) or in solution (right panel). Data are presented as the mean ± confidence interval. n = 3 for granules and n = 4 for solutions.

Follow-on studies were also conducted with an additional panel of 13 human tumor cell lines and the data are presented in Table 1. In these studies, low-serum conditions and identical analytical methods, as in the methods section, were used throughout, although the media did vary by cell line. No proliferative response was found in any cell line tested for B2A up to 10 µg/mL, although PANC-1 and HepG2 demonstrated a modest increase above that of the serum-low medium control.

Table 1.

Cell Proliferation of Various Human Tumor Cells Line Following Stimulation With B2A.^a

Name	Origin	% of control value	SD	P
HL-60	Acute promyelocytic leukemia	108.7	4.5	NS
K-562	Chronic myeloid leukemia	101.4	3.3	NS
MCF-7	Breast carcinoma	101.2	10.2	NS
SaOs-2	Osteosarcoma	96.1	12.8	NS
CaCo-2	Colon adenocarcinoma	94.4	9.7	NS
LS-174T	Colon adenocarcinoma	94.7	6.7	NS
WiDr	Colon adenocarcinoma	94.7	5.9	NS
Hep G2	Hepatocellular carcinoma	115.7	7.1	NS
AsPC-1	Pancreas adenocarcinoma	95.4	4.8	NS
Capan-1	Pancreas adenocarcinoma	80.0	7.6	NS
MIA PaCa-2	Pancreas adenocarcinoma	107.0	7.9	NS
PANC-1	Pancreas adenocarcinoma	117.5	9.5	NS
BeWo	Choriocarcinoma	91.3	7.2	NS

Abbreviation: NS, not significant; SD, standard deviation.

^a Human tumor cells were treated with 10 μg/mL of B2A for 3 or 4 days and data are presented as the average ± SD, n = 3. Statistical significance was determined by paired Student t testing using the raw data and was compared to controls.

Epithelial-Mesenchymal Transition

Epithelial-mesenchymal transition is a program of cell behavior wherein cells change shape from an epithelial to mesenchymal phenotype⁹ and increase cell motility and decrease adhesion. The EMT is a feature acquired as tumor cells become metastatic^10
–12 and is also essential for numerous normal developmental processes. During EMT, epithelial cell–cell adhesion is decreased by downregulation of E-cadherin and β-catenin¹³; additionally, molecules typically associated with the mesenchymal phenotype, such as vimentin and fibronectin, are upregulated.

B2A-treated human pancreatic PANC-1 cells did not appear to undergo EMT as evidenced by retention of the epithelial phenotype; retention of E-cadherin and β-catenin at cell-to-cell membrane junctions; and lack of fibronectin neoexpression.

Four colon cancer cell lines were evaluated in the EMT assay. B2A did not decrease the expression level of the epithelial marker E-cadherin in any cell line tested (Caco-2, LS 174T, LoVo, WiDr), nor did it cause an increased expression or redistribution of β-catenin. B2A did not increase the levels of mesenchymal cell markers vimentin or fibronectin.

Invasion

The metastatic sequence involves detachment of cells within a primary tumor, local migration and invasion of local stromal tissue extravasation, and invasion at secondary sites.¹⁴ Pharmacological signaling events that impact tumor cells invasion may, therefore, impact on cancer progression.

To test whether B2A could impact tumor cell invasion, the transwell cell model, one of the most commonly used invasion models, was used. B2A did not increase tumor cell invasion in human lung, colon, or prostate tumor cells in vitro at concentrations up to 10 µg/mL. Similarly, B2A did not increase tumor cell invasion in human breast cancer cells (MCF-7), pancreatic cancer cells (Mia PaCa-2 and PANC-1), or colon cancer cell lines (Caco-2, LS 174T, and WiDr) at 5 µg/mL.

In Vivo Effects on Human Tumor in Xenograft Models

Colon Tumors

Athymic rats were used in the evaluation and were implanted with either B2A-coated granules or sham-coated granules during posterolateral spinal fusion surgery. No animal exhibited infection or inflammation at the tumor cell inoculation site or the fusion site, and 1 week after the spinal fusion, the surgical sites in all the rats were completely healed. One animal in the B2A-coated granule group developed a palpable tumor mass at 1 week but did not grow thereafter and was excluded from the analysis. All tumors grew relatively quickly and there was no change in the shape of the growth curve (Figure 2); additionally, there was no mortality.

Figure 2.

B2A had no effect on the growth of human colon tumor (LoVo) xenografts in athymic rats. The figure shows the tumor volume at indicated time points. LoVo cells were injected subcutaneously 2 days before spine fusion surgery. B2A-coated ceramic granules mixed 1:1 with isograft bone chip (solid circles) were compared to animals with sham-coated ceramic granules mixed 1:1 with isograft bone chip surgery (open circles). The data are presented as mean number ± confidence interval.

The experiment was terminated at 4 weeks and final tumor volume and tumor weight were evaluated. The tumor volume data as well as the tumor weight data failed the normality test (Shapiro-Wilk test, P <.050); therefore, the difference between the 2 groups was analyzed by 1-way ANOVA on ranks (Kruskal-Wallis). The difference between B2A-coated granules and the uncoated granule control group was not statistically significant for either tumor volume or tumor weight (Table 2). As a percent of the uncoated control group, the median tumor volume in B2A-coated granule group was approximately 8% lower than the control group.

Table 2.

LoVo (human colon adenocarcinoma) Xenograft Tumor Volume and Tumor Weight 4 weeks After Implant of B2A-Coated Ceramic Granules or Uncoated Control Granules (Sham-Coated) During Posterolateral Spinal Arthrodesis.

Group^a	Median	25%	75%
	LoVo tumor volume in mm³
B2A-coated	1043^b	729	1167
Sham-coated	1130	913	1303
	LoVo tumor weight in mg
B2A-coated	128.0^c	106.0	172.0
Sham-coated	150.0	60.0	249.8

^an = 9 and 8 for the B2A-coated granule group and sham-coated granules, respectively.

^bNot statistically different from controls, P = .386.

^cNot statistically different from controls, P = .698.

Lung Tumors

In all, 12 rats in the B2A-coated granule group and 10 in the uncoated granule group were used in the evaluation following spinal fusion surgery. No animal exhibited infection or inflammation at the tumor cell inoculation site or the fusion site, and one week after the spinal fusion, the surgical sites in all the rats were completely healed. All tumors grew relatively slowly and the experiment was terminated at 6 weeks. There was no mortality.

The tumor volume data failed the normality test (Shapiro-Wilk test, P <.050); therefore, the difference between the 2 groups was analyzed by 1-way ANOVA on ranks (Kruskal-Wallis). The difference between B2A-coated granules and the uncoated granule control group was not statistically significant for tumor volume (Table 3). As a percent of the uncoated control group, the median tumor volume in the B2A-coated granule group was approximately 39% lower, while the tumor weight was approximately 15% lower.

Table 3.

A549 (Human Lung Carcinoma) Xenograft Tumor Volume 6 Weeks After Implant of B2A-Coated Ceramic Granules or Uncoated Control Granules (Sham-Coated) During Posterolateral Spinal Arthrodesis.

	A549 tumor volume in mm³
Group^a	Median	25%	75%
B2A-coated	87.5 ^b	32.0	162.0
Sham-coated	144.0	44.3	211.5

^an = 12 and 10 for the B2A-coated granule group and/or uncoated granules, respectively.

^bNot statistically different from controls, P = .425.

Prostate Tumors

To establish whether B2A-coated ceramic granules could accelerate primary tumor growth and/or metastasis formation in an orthotopic model of prostate cancer, a luminescent imaging model was used. In this model, PC-3M-luc cells were orthotopically implanted in the prostate, B2A-coated granules or sham-coated granules were implanted after an additional week, and then tumor progression and metastasis formation were monitored every week for 4 weeks.

One animal was found dead the next day after xenograft surgery. One animal in the sham surgery group was removed from analysis due to failure of the tumor xenograft. All the remaining animals survived for the entire study duration. There were 10 animals used in the B2A-coated granule group and 9 in the sham surgery group. There was no implant- or surgery-associated mortality.

B2A-coated ceramic granules had no effect on tumor progression, metastasis formation, or animal survival when compared to the control group (Figure 3).

Figure 3.

B2A had no effect on the growth or metastasis of human prostate tumor xenografts in severe combined immunodeficiency (SCID) mice (PC-3M-luc2). The figure shows progression of the primary tumor (panel A) and metastasis formation (panel B) based on bioluminescent imaging at indicated time points. PC-3M-luc2 cells were injected orthotopically into the prostate (asterisk) one week before surgical implant (lumbar spine, indicated by arrows) of B2A-coated ceramic granules mixed 1:1 with isograft bone chip (open circles) were compared to animals with sham surgery (solid circles). The data are presented as mean number of photons/second ± standard error of the mean (SEM).

To evaluate whether B2A could accelerate tumor growth, athymic mice bearing PC-3 human prostate tumor xenografts were treated with 3 serial bolus intraperitoneal injections of B2A each at a dose of 1 mg/kg, resulting in a cumulative dose nearly 50 times the target clinical dose of B2A-coated granules as would be used in spinal fusion surgery. There was no statistical difference (Student t test, P = .193) in PC-3 human prostate tumor weight with or without B2A treatment.

Discussion

B2A-coated ceramic granules are being investigated in combination medical devices to augment bony fusion following implant during arthrodesis surgery. The current studies, conducted in advance of pivotal clinical trials, evaluated the possibility that B2A-coated granules could enhance tumor growth.

Tumorigenesis is a multifactorial and multistep process that can be enhanced by nongenetic mechanisms such as mitogenesis and chronic inflammation (among others) as well as by genetic mechanisms such as genetic instability induced by genotoxic/cytotoxic effects.¹⁵ In the present study, to address genetic instability, one set of studies evaluated genotoxicity and cytotoxicity. The extracts of the B2A-coated granules were found to be without activity in a battery of genetic toxicology tests including a bacterial mutation assay, chromosomal aberration assay in cultured mammalian cells, and in vivo chromosomal damage assay in mice. Extracts of B2A-coated granules were evaluated consistent with their intended use as a (combination) medical device.^7,8 Similarly, extracts of the B2A-coated granules were found to be without cytotoxic activity, a feature shared with B2A based on in vitro studies. Similarly, as B2A is not genotoxic (or cytotoxic), it is unlikely that its exposure to tumor cells would instigate by additional mutations further genetic instability and would thereby not result in enhanced tumor growth potential.

There has been a perceived need to characterize the potential enhancement of tumor growth in animal models for at least some biotechnology-derived pharmaceuticals^16,17 and other pharmaceuticals ^18,19 principally those with mitogenic activity. This perceived need has also extended to orthopedic combination devices containing recombinant growth factors.^20,21 Although B2A is not derived from recombinant technology, it does have weak mitogenic activity in some, but not all, normal cells.²² Consequently, a second series of specially designed studies evaluated tumor growth enhancement. In vitro studies with human tumor cell lines demonstrated no enhanced tumor cell proliferation following exposure to B2A or B2A-coated granules. Furthermore, no enhancement of tumor growth was found in in vivo studies with human tumor xenografts (lung, colon, and prostate origin). In these studies, surgically implanted B2A-coated granules were used to mimic, to extent possible, the B2A release kinetics and B2A systemic availability as might be found in humans.

A third series of specially designed studies evaluated tumor invasion/metastasis enhancement. In vitro studies with human tumor cell lines demonstrated that neither exposure of cells to B2A or B2A-coated granules enhanced phenotypes associated with metastasis, for example increased invasiveness and increased EMT. Furthermore, no enhancement of tumor metastasis was found in in vivo studies with a human prostate tumor orthotopic model.

The results of the studies presented in this report showed no tumor growth-stimulating signal in either the in vitro tumor cell proliferation studies or the human tumor xenograft models. Additionally, there were no proliferative signals (eg, hyperplasia) found in any organ-chronic studies in rats (data not presented).

In conclusion, B2A and B2A-coated ceramic granules consistently demonstrated a lack of tumor enhancement, and, based on currently available nonclinical data, the possibility of tumor growth promotion appears unlikely. Additional larger, randomized, controlled, pivotal clinical studies designed to demonstrate safety and effectiveness of B2A-coated granules in orthopedic uses may help confirm these findings.

Footnotes

Acknowledgments

Editorial support was provided by M. C. Schneider. The work presented here was done in part in collaboration with Brent Atkinson, formerly of BioSurface Engineering Technologies, Inc.

Declaration of Conflicting Interests

The authors Zamora, Liu, Guo, and Lin were employees of BioSurface Engineering Technologies, Inc at the time the studies were conducted.

Funding

The author(s) disclose receipt of the following financial support for the research, authorship, and/or publication of this article: funded by BioSurface Engineering Technologies, Inc.

References

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