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In Vivo Hard and Soft Tissue Response of Two-Dimensional Nanoparticle Incorporated Biodegradable Polymeric Scaffolds Jason Thomas Rashkow, Yahfi Talukdar, Gaurav Lalwani, and Balaji Sitharaman ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00425 • Publication Date (Web): 09 Aug 2017 Downloaded from http://pubs.acs.org on August 15, 2017

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ACS Biomaterials Science & Engineering

In Vivo Hard and Soft Tissue Response of Two-Dimensional Nanoparticle Incorporated Biodegradable Polymeric Scaffolds Jason T Rashkow1, Yahfi Talukdar1, Gaurav Lalwani1, and Balaji Sitharaman1*

1

Department of Biomedical Engineering, Stony Brook University, Stony Brook, NY 11794-5281

*Correspondence Balaji Sitharaman, Ph.D. Associate Professor Department of Biomedical Engineering Bioengineering Building, Rm 115 Stony Brook University, Stony Brook, NY 11794-5281 Tel: +1-631-632-1810 Fax: +1-631-632-8577 Email: [email protected]

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Abstract Current efforts in the design of bone tissue engineering scaffolds have focused on harnessing the physicochemical properties of two-dimensional organic and inorganic nanoparticles to improve bulk and surface properties of biodegradable polymers. Herein, we investigate the hard and soft tissue in vivo biocompatibility of two such constructs; 90% porous poly(lactic-co-glycolic acid) (PLGA) nanocomposite scaffolds incorporated with 0.2 wt% graphene oxide nanoplatelets (GONPs) or molybdenum disulfide nanoplatelets (MSNPs). Scaffolds were implanted in a noncritical sized monocortical defect in the tibia or subcutaneously on the dorsum of a rat model for two or six weeks. Hard and soft tissue in vivo biocompatibility of the nanoparticle reinforced scaffolds was comparable to PLGA control. In addition, two weeks after implantation, significantly less bone growth (~35%) was observed for the PLGA group compared to the empty defect group; not observed for the experimental groups which showed 20% and 15% greater bone growth compared to the PLGA group. This may indicate that the nanoparticles do play a role in assisting bone regeneration. Taken together, the results suggest that scaffolds incorporated with GONPs or MSNPs show promise for bone tissue engineering applications.

Keywords Nanoparticles, Scaffold, Biocompatibility, Tissue Engineering


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Introduction The distinct physiochemical properties of layered two-dimensional (2D) carbon nanoparticles (e.g. graphene) and inorganic nanoparticles (e.g. molybdenum disulfide) significantly improve the bulk and surface properties of biocompatible biodegradable polymers employed for tissue engineering applications.1-3 Few studies have investigated the in vivo biocompatibility of these nanoparticle-incorporated

polymeric matrices.4-7

All

these studies have focused

on

nanocomposites incorporated with graphene nanoparticles known as graphene oxide or graphene oxide nanoplatelets (GONPs) implanted in soft tissues.4-7 Kanayama et al. examined the soft tissue (subcutaneous) biocompatibility of GONP and reduced-GONP coated collagen scaffolds in a rat model after being implanted for 10 days.5 Zhou et al. reported the inflammatory response by microglia and astrocytes after implanting electrospun poly(ε-caprolactone)

microfiber

scaffolds coated with colloidal graphene into neural tissues (striatum or into the subventricular zone) of adult rats for 7 weeks.7 Wang et al. investigated the addition of graphene to polyethylene-terephthalate for use in an extra-articular graft-to-bone healing procedure in New Zealand rabbits for 4, 8, and 12 weeks.6 Finally, Duan et al. examined the biocompatibility of mesenchymal stem cell (MSC) seeded graphene reinforced poly(L-lactic acid) scaffolds implanted intramuscularly in nude mice for 2, 4, and 8 weeks.4 To the best of our knowledge; no in vivo investigation has examined and compared the short and long term soft and hard tissue responses of porous polymeric biodegradable scaffolds incorporated with layered 2D carbon or inorganic nanoparticles.

Herein, we report, in rodents, the short (2 weeks) and long (6 weeks) term hard and soft tissue biocompatibility of porous poly(lactic-co-glycolic acid) (PLGA) scaffolds incorporated with



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layered 2D carbon and inorganic nanoparticles (graphene oxide nanoplatelets (GONPs) and molybdenum disulfide nanoplatelets (MSNPs).


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Experimental Section

Methods Nanoparticle Synthesis and Characterization GONPs were synthesized from graphite flakes using a modified Hummer's method.8 MSNPs were synthesized through a high temperature reaction between MoO3 and sulfur powder (SigmaAldrich, St. Louis, MO, USA).9 Nanoparticles were characterized by high resolution transmission electron microscopy (HRTEM) and Raman spectroscopy. For HRTEM, nanoparticles were dispersed in water and ethanol (1:1 solution) by sonication. Dispersion was followed by ultracentrifugation and the supernatant was drop cast on a lacey carbon grid (300 mesh size, copper support, Ted Pella, Redding, CA, USA). HRTEM imaging was performed using a JOEL 2100F high-resolution analytical transmission electron microscope (Peabody, MA, USA) with an accelerating voltage of 200 kV at the Center for Functional Nanomaterials, Brookhaven National Laboratory, New York. Raman spectra were recorded between 50–3750 cm−1 using WITec alpha300R Micro-Imaging Raman Spectrometer (Ulm, DE) equipped with a 532 nm Nd-YAG excitation laser at room temperature.

Scaffold Fabrication PLGA (50:50 polylactic:glycolic acid, MW = 111,500 Da; Polysciences, Warrington, PA, USA) was dissolved in chloroform (Sigma-Aldrich). To achieve loading of 0.2 wt% GONPs or MSNPs, nanoparticles were dispersed in the polymer solution by heating to 60 °C for one hour followed by sonication for 30 minutes. PLGA without nanoparticles was used as control. 90% porous scaffolds were prepared by adding polymer/nanoparticle solution to NaCl porogen (size



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range: 200–500 µm) at a ratio leading to desired porosity in the final scaffolds and mixed thoroughly to create a paste. Scaffold porosity was calculated using equation (1).10 (1) (%) =

Where ε is the apparent porosity of the scaffold, VNaCl is the volume of NaCl porogen, and VNC is the volume of the nanocomposite. Volumes were calculated based on the theoretical density of NaCl = 2.16 g/cm3 and PLGA nanocomposites = 1.34 g/cm3.

The composite-porogen paste was pressed into cylindrical Teflon molds with diameter of 4 mm and depth of 4 mm and left for 48 hours to evaporate off residual chloroform. Once dry, samples were pressed out of the molds, and the porogen was leached through submersion in agitated deionized water for 3 days followed by vacuum drying. Prior to implantation, all scaffolds were be submerged in 70% ethanol for 30 minutes and exposed to UV light for another 30 minutes. For BMP-2 caffolds, immediately before implantation, 20 PLGA scaffolds were saturated with a solution containing 30 µg/ml BNP-2 (1.89 µg BMP-2 per 62.83 mm3 scaffold volume, Novoprotein, Summit, NJ, USA).

Animals All animal procedures were approved by Stony Brook University’s Institutional Animal Care and Use Committee (IACUC). 100 Male Wistar rats (12 weeks old, weight = 350-400 g, Charles River, Wilmington, MA, USA) were used to investigate nanoparticle reinforced scaffold hard and soft tissue biocompatibility. Throughout the study, all rats were allowed to roam freely and were provided with standard chow diet and water ad libitum. All rats were kept on a 12 hour


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light / dark cycle, where the lights will be turned on during the day and off at night. When time points were reached, the animals were euthanized using carbon dioxide inhalation.

Implantations For hard tissue biocompatibility, the animals were divided into 5 groups and two time points; 2 and 6 weeks (10 rats per group) (see Table 1). For soft tissue biocompatibility, 60 of the 100 animals also had scaffolds implanted under the skin. Animals were anesthetized for surgery with Isoflurane (1-2.5%, IsoThesia, Henry Shein, Melville, NY, USA) in O2 by inhalation. After the surgery, animals were administered analgesics to reduce pain (Buprenorphine 0.015mg/ml, dose 0.01 mg/kg). Table 1. Groups for soft and hard tissue analysis. Animals per group Hard Tissue Groups 2 Weeks 6 Weeks Empty defect 10 10 PLGA scaffold with BMP-2 10 10 PLGA scaffold 10 10 PLGA scaffold reinforced with GONP 10 10 PLGA scaffold reinforced with MSNP 10 10

Animals per group Soft Tissue 2 Weeks 6 Weeks

10 10 10

10 10 10

Hard tissue Implantation Non-critical sized monocortical plug defects were created unilaterally at the anteromedial surface of the tibia. An incision was made in the skin exposing the proximal tibia, the location of the hole was marked on the bone based on the distance from the joint. The hole (4 mm in diameter and depth) was made using a hand drill with a trephine burr and the scaffold was pressed into the hole. Finally, the surrounding area was washed with saline and the muscle and skin layers were sutured back into place.



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Soft Tissue Implantation and Blood Collection Control and nanocomposite scaffolds were implanted within subcutaneous pockets in the dorsum of the rats. The dorsum of the rat was shaved, washed and disinfected with povidone-iodine. Two longitudinal incisions of 1 cm were made through the full thickness of the skin, one on each side of the spinal column. The subcutaneous pockets were created laterally to the incision by blunt dissection and the scaffolds were placed in the pockets. Once implanted, the area was irrigated with saline and the skin was sutured.

In addition to scaffold implantation, levels of proinflammatory cytokine TNF-α in animal plasma were determined using an enzyme-linked immunoassay (ELISA) kit from R&D Systems (Minneapolis, USA). Prior to surgery and at the 2 week end point, 1 ml of blood was collected from the rats through an intravenous catheter that was inserted into the tail vein. Blood was centrifuged at 1000 rpm for 20 minutes and stored at -80 °C until analysis.

Microcomputed Tomography (Micro-CT) Evaluation At the end of each time point, the animals were euthanized by 100% CO2 inhalation. Bones with defects were collected and fixed in 10% formalin for 24 hours, then washed with PBS and stored in 70% ethanol until analysis. Bone volume fraction (BV/TV) and bone density were obtained with a micro-CT system (µCT-40, Scanco, PA, USA). The boundary of the drilled hole was contoured as region of interest for 150 slices of each scaffold. The CT system was operated at a voltage of 55 kVp, current of 145 A, giving a resolution of 20 µm3. Evaluations were done using the Scanco evaluation software with a threshold of 220, Gaussian noise filter of 1.2, and support of 2.


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Histological Analysis After Micro-CT, five samples from each group were decalcified and embedded in paraffin. The tissues were cross-sectioned at 5 µm thickness in the longitudinal parallel direction. Mineralized bone matrix and osteoid were evaluated by Masson’s trichrome staining. Histologic analysis was used as a second method to quantitatively measure bone ingrowth at 2 and 6 weeks and multinucleated giant cell response at 2 weeks. Using Osteomeasure software (OsteoMetrics, GA, USA), bone ingrowth was calculated by selecting the area of the scaffold as the region of interest

(ROI) and outlining bone growth within the ROI as the bone ingrowth area (BIA) in a histological section of each sample. Using these values, percentage bone ingrowth area was calculated using equation (2). (2) % Bone ingrowth = (BIA/ROI x 100%) For multinucleated giant cell response, four regions within the defect area were selected and giant cells were counted within the regions.

Histological Scoring Analysis Analysis of the hard tissue response was graded using an adapted scale presented in Table 2.11,12 After the animals were euthanized at 2 and 6 weeks, scaffolds and surrounding skin were collected for histological analysis. Samples were immediately placed in 10% formalin for 24 hours. The samples were then rinsed with PBS rinse, and kept in 70% ethanol until histological processing. Three samples from each group were embedded in paraffin and sectioned at a thickness of 5 µm. Staining of the sections was completed using hematoxylin and eosin (H & E) and alizarin red S. H & E stained sections were used for histological grading of capsule thickness and interstitium tissue quality. Analyses of tissue architecture were completed though a 9 ACS Paragon Plus Environment


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previously used scoring system presented in Tables 3 and 4.11,12 Capsule thickness was analyzed by measuring the distance from the scaffold surface to the adjacent muscle tissue in four locations around the scaffold. Alizarin red S stained sections were used to determine if ectopic bone formation had occurred within the tissue.

Table 2. Histological grading scale for hard tissue response. Description Tissue in pores is mostly bone Tissue is mostly bone with some fibrous tissue and/or a few inflammatory response elements Tissue in pores consists of some bone with fibrous tissue and/or a few inflammatory response elements Tissue in pores is mostly fibrous tissue (with or without bone) and young fibroblasts invading the space with giant cells present Tissue in pores consists mostly of inflammatory cells and connective tissue components in between (with or without bone) OR the majority of the pores are empty or filled with fluid Tissue in pores is dense and exclusively of inflammatory type (no bone present)

Score 5 4 3 2 1 0

Table 3. Capsule Thickness Score Description Score 1–4 cell layers 4 5–9 cell layers 3 10–30 cell layers 2 > 30 cell layers 1 Not applicable 0

Table 4. Histological grading scale for capsule interstitium quality. Description Tissue in interstitium is fibrous, mature, not dense, resembling connective or fat tissue in the non-injured regions Tissue in interstitium shows blood vessels and young fibroblasts invading the spaces, few giant cells may be present Tissue in interstitium show giant cells and other inflammatory cells in abundance but connective tissue components in between Tissue in interstitium is dense and exclusively of inflammatory type Implant cannot be evaluated because of problems that may not only be related to the materials to be tested

Score 4 3 2 1 0 10

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Statistics All graphs are presented as average ± standard deviation. All data were analyzed for significance by analysis of variance (ANOVA) with Tukey post hoc. Differences with p < 0.05 were considered significant.



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Results and Discussion The overall objective of this study was to investigate the short- and long-term hard and soft tissue biocompatibility of biodegradable polymeric scaffolds-incorporated two-dimensional layered nanoparticles. The composition of nanoparticle will affect a scaffold’s surface properties, and consequently, influence the scaffold’s interaction with surrounding tissue. The responses of these interactions may directly affect the scaffold’s biocompatibility. Therefore, we employed 2D layered nanoparticles that were isostructural but of different composition – the widelystudied carbon nanoparticle graphene oxide nanoplatelets (GONPs),1 and inorganic nanoparticle molybdenum disulfide nanoplatelets (MSNPs, an isostructural analog of graphene).13 These nanoparticles were also chosen based on previous physiochemical and in vitro cytocompatibility studies.14,15 Those studies indicated that GONPs or MSNPs enhance the bulk (mechanical) and surface properties of biodegradable biocompatible polymers, and thus are promising candidates to improve the polymer’s functional properties. Indeed, the addition of 0.2 wt% GONPs or MSNPs to polypropylene fumarate was found to increase the compressive modulus by ~40% and ~50%; and compressive yield strength by ~40% and ~53%, respectively compared to polymer alone.14 PLGA 50:50 was selected for this study due to reported short degradation time to match the fast healing time of rats.16 PLGA scaffolds loaded with 0.2 wt% nanoparticles were chosen based on prior in vitro cytotoxicity screening studies which showed that this scaffold configuration did not elicit any adverse effects on cells.3 Additionally, if the nanoparticles loaded at 0.2 wt% are released from a porous scaffold with volume 10 cm3, the total nanoparticle concentration is ~ 20 µg/ml. This concentration was previously found to be potentially safe for adult stem cells.9,17,18 Scaffolds with porosity of 90% with pore size in the range of 200-500 µm were fabricated since previous work has shown that a minimum pore size of 100 µm is necessary


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for cellular infiltration and transport of nutrients and waste, however scaffolds with pores of 300 µm show greater vascularization and osteogenesis.19

Nanoparticles and Nanocomposites Representative HRTEM images of GONPs and MSNPs are shown in Figure 1 A and B. GONPs (Figure 1 A), were flat disk-like sheets with diameters of 100-200 nm. MSNPs (Figure 1 B) showed similar features as GONPs - disk-shape and diameters in the range of 40-90 nm. Representative Raman spectra of the two nanoparticles are presented in Figure 1 C. The spectra for GONP (Figure 1 C [i]) showed peaks at 1352 cm-1 (d-band) and 1602 cm-1 (G-band). The spectra of MSNPs (Figure 1 C [ii]) showed peaks at 370 cm-1, 398 cm-1 and 476 cm-1. The Gband is indicative of pristine sp2 carbon network, and the D-band, structural defects in the graphene sheet.20 The amount of defects in the structure is inferred from the ratio of the intensity of D- to G-bands.21 The ID/IG for GONP was 1.03; much higher compared to pristine carbon structures.8 The bands observed in the MSNP Raman spectra are the two central bands for dichalcogenide materials. The band observed at 370 cm-1 represents the E2g1 Raman active mode indicating in-plane vibration. The second band at 398 cm-1 represents the A1g Raman active mode for the out-of-plane vibrations between the molybdenum and sulfur atoms.22,23 The small peak at 476 cm-1 represents small amounts of unreacted MoO3.



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Figure 1. Representative HRTEM images of (A) GONPs and (B) MSNPs. (C) Raman spectra of GONPs (i) and MSNPS (ii).

Table 1 lists group and number of animals per group for the hard and soft tissue biocompatibility. The porous scaffolds were implanted into a 4 x 4 mm3 defect in a rat tibia or subcutaneously on the rat dorsum. The non-critical sized tibial defect model was chosen since it minimizes variation between animals as compared to segmental defect which requires a more invasive surgery.24 Moreover, as the main objective of this study was to investigate the biocompatibility of the nanocomposite scaffolds, this outcome was still observable without a critical sized defect. For the hard tissue response studies, PLGA scaffolds soaked with BMP-2


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were used as positive control due to BMP-2’s proven promotion of bone formation and current clinical use. The concentration of 30 µg/ml was used as this concentration has been shown to fuse a segmental defect in rats without causing adverse effects observed at higher doses.25

Animal Surgery All animals that underwent surgery recovered after the surgical procedures and remained in good health during the duration of the study. No signs of wound complications or abnormal behavior were noted in any of the animals. Additionally, proinflammatory cytokine TNF-α plasma levels were below the detection threshold (5 pg/ml) of the ELISA assay for blood collected at baseline as well as 2 weeks after surgery (data not shown).

MicroCT and Histomorphometric Analysis Figure 2 shows the micro-CT results of the hard tissue response at 2 and 6 weeks. Qualitatively, at both the time points, representative three-dimensional reconstructions (Figure 2 A) of the noncritical sized tibial defects show a smaller defect size in the empty defect compared to groups that received the scaffolds. No discernible differences in bone formation were obvious across all groups by 6 weeks. Quantitatively, at two weeks, the bone formation in defects with PLGA scaffolds (Figure 2 B) showed significantly less healing (~35%) compared to empty defects. Bone volume fraction in GONP and MSNP groups were 20% and 27% lower than empty defect group and 20% and 15% greater compared to the PLGA group, respectively. However, these differences were statistically insignificant. At 2 weeks, bone formation in defects implanted with BMP-2, GONP, or MSNP scaffolds showed similar healing to the empty defect group. After 6



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weeks, there were no significant differences in bone volume fraction (Figure 2 B) between all the groups.

Bone density, measured as mass of hydroxyapatite per volume of bone, provides a good indication of bone strength. At 2 weeks, there were no significant differences in bone density across all groups (Figure 2 C). However at 6 weeks, bone in the empty defects was significantly higher compared to defects implanted with scaffolds (Figure 2 C), and no statistically significant differences were noted between the groups implanted with scaffolds. A possible explanation for the discrepancy between empty defect and scaffold groups could be due to differences in bone regeneration rates. The empty defect is not a critical-sized defect. Thus, without the physical barrier created by the scaffolds which could delay the regenerative process,26 the empty defects allow greater bone formation and consequently, higher bone density compared to the scaffold groups.


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Figure 2. MicroCT results. (A) Representative three-dimensional reconstructions of monocortical defects 2 weeks or 6 weeks after surgery, treated with: empty defect, PLGA, BMP2, GONP, or MSNP. Scale bars are 1 mm. (B) Quantitative microCT evaluations for bone volume fraction (BV/TV) 2 weeks or 6 weeks after surgery. (C) Measurement of bone density within the defect site 2 weeks or 6 weeks after surgery. Data is presented as mean +/- standard deviation (n=10). Statistical significance was determined by ANOVA with Tukey post hoc and p

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