Investigation of an injectable gold nanoparticle extracellular matrix

Abstract

Post-traumatic osteoarthritis (PTOA) is a progressive articular degenerative disease that degrades articular cartilage and stimulates apoptosis in chondrocyte cells. An injectable decellularized, extracellular matrix (ECM) scaffold, that might be able to combat the effects of PTOA, was developed where the ECM was conjugated with 20 nm gold nanoparticles (AuNP) and supplemented with curcumin and hyaluronic acid (HA). Porcine diaphragm ECM was decellularized and homogenized; AuNPs were conjugated using chemical crosslinking followed by mixing with curcumin and/or HA. Injection force testing and scanning electron microscopy with energy-dispersive X-ray spectroscopy were utilized to characterize the ECM scaffolds. In vitro testing with L929 murine fibroblasts, equine synovial fibroblasts, and Human Chondrocytes were used to determine biocompatibility, reactive oxygen species (ROS) reduction, and chondroprotective ability. The results demonstrated that conjugation of 20 nm AuNPs to the ECM was successful without significantly altering the physical properties as noted in the low injection force. In vitro work provided evidence of biocompatibility with a propensity to reduce intracellular ROS and an ability to mitigate apoptosis of chondrocyte cells stimulated with IL-1β, a known apoptosis inducing cytokine. It was concluded that an injectable AuNP-ECM may have the ability to mitigate inflammation and apoptosis.

Keywords

extracellular matrix gold nanoparticles anti-inflammatory agents homogenized tissue

Introduction

The use of extracellular matrix (ECM) derived scaffolds over synthetic biomaterials for therapeutic applications is becoming increasing popular due in part to ECM’s inherent biomechanical and biochemical cues. These natural cues influence the cellular microenvironment and play an important role in cell cycle progression, cell fate decision, and migration of cells.^1,2 The ECM, in particular, is inherently laden with cytokines, growth factors, and integrins that can stimulate recruitment and migration of cells for repair and regeneration in the region of implementation.^3–7

The benefits of the decellularized ECM can be expounded through homogenization. Homogenization of decellularized ECM allows more flexibility in terms of therapeutic applications, such as being amendable for injection, while preserving the natural microcellular environments.⁸ Homogenized extracellular matrix (hECM) can then conform to the shape of its environment. For example, Saleh et al.⁹ developed a homogenized liver ECM. By perfusing and subsequently conjugating homogenized liver ECM into a decellularized liver, they were able to achieve an increased cellular infiltration and an increased population of hepatic regenerative cells relative to a decellularized liver alone in a rat model. Another study by Choi et al.¹⁰ utilized a lyophilized ECM derived from adipose tissue for injectable delivery cell for applications in adipose tissue engineering. In a mouse study using the homogenized ECM with adipose derived stem cells, they were able to demonstrate neovascularization along with adipogenesis and accumulated lipid droplets.

Our group investigated an injectable hECM for the possible treatment of post-traumatic osteoarthritis (PTOA). Post-traumatic osteoarthritis is a disease wherein a traumatic event propagates the degradation of articular cartilage over time and thus may result in bone-on-bone articulation.¹¹ Articular cartilage is the cartilage found on the end of bones in the joint space to aid articulation of joints and ease the impact placed on the joint. To date, no cure has been identified to mitigate the degradation of articular cartilage; temporary therapies that ease pain and aid articulation are utilized today and include physical therapy and non-steroidal anti-inflammatory drugs. The progression of PTOA begins with immediate necrosis of chondrocyte cells after a direct, traumatic impact. The chondrocyte cells surrounding the impacted region are also affected, resulting in dysregulated and hypertrophic release of cytokines like interleukin-1β (IL-1β) that further dysregulate the cells, degrade the articular cartilage matrix, and eventually lead to apoptosis of cells.^12–16 Because of the detrimental effects, there has been much research on finding more effective treatment methods to reduce inflammation found in PTOA.^17–22

One treatment method that has been researched is the use of hyaluronic acid (HA). Hyaluronic acid is a naturally occurring glycosaminoglycan found in the body. It can increase synovial fluid viscosity and aid in compression absorption of the articular cartilage by drawing water into the ECM.²³ While HA is well known for its lubricating ability, it has also been shown to promote ECM regeneration and to influence the inflammatory response.²⁴ For example, in a recent study, Barakat et al.²⁵ performed a randomized prospective pilot study on HA injections in intra-articular knee fractures. They concluded that HA played a direct role in the acute phase of the inflammatory process and may result in reduce pain for the patient.

Another possible anti-inflammatory treatment method is the use of curcumin, which is known for its anti-inflammatory and free radical scavenging ability.²⁶ Curcumin is a naturally derived component of turmeric that has been used in many therapeutic applications.^27–29 For example, research performed by Shakibaei et al.³⁰ demonstrated curcumin’s interaction with osteoarthritic human chondrocyte cells. They observed a downregulation of known osteoarthritic pathway NF-kB through incubation with curcumin with IL-1β and tumor necrosis factor-alpha stimulated chondrocyte cells.

A possible anti-inflammatory agent that our group has investigated is gold nanoparticles (AuNPs). AuNPs possess a number of advantageous properties. AuNPs have shown a reduction in inflammation through free radical scavenging.^8,31–33 Research has also shown encouragement of cellular migration and attachment, which is believed to be achieved through the higher surface energy of the nanoparticles.³⁴ Also, manipulation of the degradation rate of the scaffold may be possible due to conjugated AuNPs blocking collagenase binding sites and thus providing a longer lasting matrix.³⁵

This paper described the development of decellularized hECM supplemented with curcumin, HA, and conjugated AuNPs. Material characteristics such as injectability of the hECM were determined. Scanning electron microscopy along with energy dispersive X-ray spectroscopy was utilized to verify evidence of AuNP conjugation. In vitro analysis was performed with murine fibroblast cells, equine synovial fibroblast cells, and human chondrocyte cells to determine the biocompatibility, free radical scavenging, and chondroprotective ability of the supplemented hECM.

Materials and methods

Harvesting tissue and ECM decellularization

Porcine diaphragms were harvested from the University of Missouri School of Medicine after euthanasia and subsequently placed in a Tris buffer solution. Following our established protocol, the diaphragms were then decellularized by placing them in a 1% (v/v) tributyl phosphate Tris buffer solution and agitated on a shaker table at 225 r/min for 24 h³⁶ After the initial wash, two 24-h washes in DI H₂O followed by a 24-h wash in 70% (v/v) ethyl alcohol were performed. All decellularized ECMs were then stored in 70% (v/v) ethyl alcohol at 4°C.

ECM homogenization

A blade homogenizer (IKA T10 Basic, Wilmington, NC) was utilized for ECM homogenization. Diaphragm ECM was manually cut into small pieces using a scalpel to aid the blade homogenization. Approximately 1 g of wet ECM was added to 15 mL of phosphate buffered saline (PBS). The ECM/PBS solution was placed on ice 15 min prior to homogenization and remained on ice during homogenization to keep the solution cool. To homogenize, the blade homogenizer was set to setting five and immersed into the ECM/PBS solution for 1 min and taken out of the solution for 1 min. This was cycled five times to prepare an injectable hECM solution. The hECM was stored at 4°C for later testing.

Preparation of homogenized construct

The 20 nm AuNPs were purchased from Ted Pella (Redding, CA). AuNPs are at a stock concentration of 7.0 × 10¹¹ particles/ml. In this work, this stock concentration is considered a “1X” concentration with “4X” being four times the amount of AuNPs per mL. Prior to conjugation, AuNPs were first functionalized with 15 μM 2-mercaptoethylamine (MEA). A 1-ethyl-3-[3-dimethylainopropyl] carbodiimide (EDC)/N-hydroxysuccinimide (NHS) crosslinker was utilized to conjugate the functionalized AuNPs to the hECM. To prepare the crosslinking solution, 5 mM NHS was dissolved in dimethylformamide and 2 mM EDC was dissolved in a 0.1 M 2-(N-Morpholino) ethanesulfonic acid (MES) in 0.5 M NaCl. The hECM was added to the crosslinking solution along with functionalized AuNPs and placed on a shaker table at 225 r/min for 1 h. After 1 h, the ECM constructs were washed with PBS three times by centrifugation at 5000 r/min.

The addition of either curcumin or HA was performed after conjugating the AuNPs to the hECM via mixing. Curcumin was purchased from Sigma Aldrich (St. Louis, MO) and HA was purchased from Lifecore Biomedical (MW = 700 kDa, Chaska, MN). Hyaluronic acid was rehydrated from a lyophilized powder with sterile PBS to their respective concentrations. A 15% (w/v) HA was utilized in all cell studies. Curcumin was dissolved in 1 N sodium hydroxide (NaOH) and a 4% (w/v) curcumin concentration was used in the cell work unless otherwise stated.

Sterilization

Samples were sterilized using a 0.1% (v/v) peracetic acid solution with 1 M NaCl. Samples were placed on a shaker table at 225 r/min for 24 h. After 24 h, the samples were centrifuged at 5000 r/min for 5 min to remove the supernatant and washed with sterile PBS three times.

Scanning electron microscopy

A FEI Quanta 600 FEG Environmental scanning electron microscope (SEM) and energy-dispersive X-ray spectroscopy (EDS) (Hillsboro, OR) was utilized for imaging and elemental analysis. Samples were placed in a fixative solution provided by the University of Missouri Electron Microscopy Core of 100 nM sodium cacodylate, 2% glutaraldehyde, and 2% paraformaldehyde overnight and rinsed three times with H₂O. Critical point drying was performed on specified samples. Samples were placed on carbon tape.

Injection force

An Instron 5900R Universal Testing machine (Norwood, MA) was utilized in compression mode to determine maximum extrusion force of hECM through a 22-gauge cannula. A constant rate of displacement (0.167 mm/s) was used to determine force of extrusion for 30 mm intervals. Force was determined every 0.1 s.

Cell culture preparation

L929 murine fibroblast cells (ATCC, Manassas, VA) were cultured in Eagle’s minimum essential medium (EMEM, ATCC, Manassas, VA) with 10% (v/v) horse serum (ATCC, Manassas, VA) and 200 U/mL penicillin streptomycin (ATCC, Manassas, VA) at 37°C with 5% CO₂. Assays were performed up to 20 subcultivations with subculturing performed weekly. Media was exchanged every 3 days when performing an assay. All culturing of cells were performed under sterile conditions using a biologic safety cabinet.

Human Chondrocytes (HCH) cells (Promocell GmbH, Heidelberg, DE) were cultured in human chondrocyte media (Promocell GmbH, Heidelberg, DE) supplemented with 0.1 mL/mL fetal calf serum (Promocell GmbH, Heidelberg, DE) cultured at 37°C with 5% CO₂. Assays were performed up to 20 subcultivations with subculturing performed weekly. Media was exchanged every 3 days when performing an assay. All culturing of cells were performed under sterile conditions using a biologic safety cabinet.

Equine synovial fibroblast cells provided from D.B. Fox’s lab group at the University of Missouri were cultured at 37°C with 5% CO₂ with DMEM supplemented with 10% fetal bovine serum, 0.008% Hepe’s buffer, 0.008% non-essential amino acids, 0.002% penicillin 100 I.U./mL streptomycin 100 μg/mL, amphoterocin B 25 μg/mL, 0.002% L-ascorbate, and 0.01% L-glutamine. Cells were assayed at their fourth passage. All culturing of cells were performed under sterile conditions using a biologic safety cabinet.

Cell viability assay

A WST-1 assay kit (Roche, Indianapolis, IN) was utilized to determine cellular viability of both L929 murine fibroblast and HCH cells. Approximately 3 × 10⁴ cells/wells were added to a 96-well plate for culture. Cells were incubated for 24 h prior to addition of hECM. The 50 μL centrifuged pellets of hECM were incubated in relevant cellular media 24 h prior to addition with cells. Media was exchanged every 3 days. The WST-1 reagent was added to each well per manufacture’s protocol and incubated for 4 h. A 450 nm absorbance value with a cutoff of 650 nm was evaluated using a Cytation 5 (BioTek, Winooski, VT). Table 1 details the hECM formulations tested.

Table 1.

Listing of the hECM samples and cell types for the WST-1 assays.

	L929 fibroblasts	HCH
Control (hECM only)	Y	Y
1×AuNPs	Y	Y
4×AuNPs	Y	Y
4×AuNPs–2% curcumin	Y	N
4×AuNPs–4% curcumin	Y	Y
4×AuNPs–6% curcumin	Y	N
1×AuNPs–4% curcumin	N	Y
15% HA	N	Y
4× AuNPs–15% HA	N	Y

hECM: homogenized extracellular matrix; HA: hyaluronic acid; AuNPs: gold nanoparticles; HCH: Human Chondrocytes.

ROS assay

An OxiSelect ROS assay kit (Cell Biolabs, San Diego, CA) was used to determine ROS activity per sample group with L929 fibroblast, synovial fibroblast, and HCH cells. Approximately 3 × 10⁴ cells/wells were plated onto a 96-well plate for culture with media being changed every 3 days. Cells were incubated for 24 h to become confluent. After 24 h, cells were washed with Dulbecco's phosphate-buffered saline (DPBS) two times and then 1X 2′, 7′-Dichlorodihydrofluorescin diacetate (DCF-DA) diluted in media was added to each well for 1 h. Cells were then washed with DPBS two times to remove the DCF-DA. The 50 μL centrifuged pellets of hECM were incubated in relevant cellular media 24 h prior to addition with cells. Cell media and hECM was then added to the well plate. The hECM and cells were incubated together for 12 h. After 12 h, cells were lysed using a cell lysis buffer provided in the assay kit. A Cytation 5 (BioTek, Winooski, VT) was utilized to determine the fluorescent intensity at 480 nm excitation and 530 nm emission. The concentration of DCF was determined by linear regression using a provided DCF standard. Table 2 details the hECM formulations tested.

Table 2.

Listing of the hECM samples and cell types for the ROS assays.

	L929 fibroblasts	Synovial fibroblasts	HCH
Control (hECM only)	Y	Y	Y
1×AuNPs	Y	N	N
4×AuNPs	Y	Y	Y
1×AuNPs–4% curcumin	Y	N	N
4×AuNPs–4% curcumin	Y	N	Y
4% curcumin	N	N	Y
15% HA	N	N	Y
4% curcumin–15% HA	N	N	Y
4×AuNPs–15% HA	N	N	Y
4×AuNPs–4% curcumin	N	N	Y
4×AuNPs–4% curcumin–15% HA	N	N	Y

hECM: homogenized extracellular matrix; HA: hyaluronic acid; AuNPs: gold nanoparticles; HCH: Human Chondrocytes.

Apoptosis assay

An HT TiterTACS assay kit (Trevigen, Gaithersburg, MD) was used to determine apoptosis of HCH cells under stimulation of IL-1β. Approximately 4 × 10⁴ cells/wells were incubated for 24 h in a 96-well plate to become confluent. Cells were then exposed to 10 ng/mL IL-1β for 24 h. Cells were then washed twice with DPBS and fixed with 3.7% (v/v) formaldehyde at room temperature for 7 min. Cells were washed with DPBS twice and incubated for 20 min in methanol, washed with DPBS and incubated with proteinase k for 15 min. Samples were washed with dH₂O; then TACS-nuclease was added to positive control groups or experimental groups and was incubated for 30 min. H₂O₂ was added for 5 min to quench endogenous peroxidase activity and washed with dH₂O. Cells were then labeled with 1× TdT labeling buffer for 5 min and then incubated in the cell incubator for 1 h with the provided labeling reaction mix. After 1 h, a 1× TdT stop buffer was added for 5 min at room temperature. Samples were washed with PBS twice. Finally, cells were incubated with Strep-HRP and TACS-Sapphire for 10 min and 30 min, respectively, at room temperature and washed with 0.1% (v/v) tween-20/PBS after the Strep-HRP incubation. A 0.2 N HCl was added, and samples were measured with an absorbance of 450 nm with a 650 nm cutoff using a Cytation 5 (BioTek, Winooski, VT).

Statistical analysis

Statistical analysis was performed using GraphPad Prism 8 software. A one way analysis of variance with pairwise Tukey test was performed with p ≤ 0.05 unless denoted otherwise in figure description. The mean with error bars representing standard deviation was utilized in this work.

Results

Electron microscopy

Conjugation of 4× 20 nm AuNPs to hECM was analyzed using SEM and EDS analysis techniques. AuNPs conjugated to hECM and AuNPs conjugated to hECM mixed with 6% curcumin were investigated to determine if the AuNPs were crosslinked to the hECM. Figure 1(a) is an image of the AuNPs-hECM sample displaying evidence of the ECMs porous microstructure, which was maintained through the process of homogenization and conjugation of AuNPs. The sample in Figure 1(a) was prepared using critical point drying. Figure 1(b) is a backscattered image displaying “white spots” indicative of AuNP. The inserted red box is the spot utilized for EDS analysis, which is shown in Figure 1(c). There is a clear peak of Au at 2.120 keV confirming the presence of AuNPs. Similarly, for the AuNP-hECM mixed with curcumin, Figure 2(a) shows AuNPs scattered throughout the ECM along with Figure 2(b), the backscatter image providing a clearer view of the AuNPs in the ECM. EDS analysis confirmed the presence of Au peak at 2.120 keV (image not shown). Figure 2(c) is a lower magnification displaying the overall structure of the homogenized sample in a hydrated state.

Injection force study

To determine injectability of the hECM, maximum compressive force was analyzed. Homogenized extracellular matrix was added to various concentrations of HA to determine the effect of HA on the injectability. The data is summarized in Table 3. All concentrations of HA were well below 1 N of force required to inject. The sample that required the most force was the PBS sample with no HA which required 0.75 N. The sample that required the least force was the 25% HA sample.

Table 3.

Max extrusion force data for hECM supplemented with multiple concentrations of HA ejected through a 22-gauge cannula.

	Max compressive force (N)	Standard deviation (±)
Control	0.75	0.07
10% HA	0.70	0.06
15% HA	0.60	0.06
20% HA	0.63	0.11
25% HA	0.57	0.24

WST-1 assays

L929 Fibroblasts—A WST-1 cellular viability study was used to determine how the murine L929 fibroblast cells interacted with the hECM samples over a 3-day time period. Results as shown in Figure 3 demonstrated that the addition of AuNPs to the hECM increased cellular activity. The same trend was observed with the addition of curcumin–AuNPs, which displayed an increase in the overall viability.

Human chondrocytes—A cell viability study was performed to determine how the homogenized constructs interacted with human chondrocyte cells. The results are shown in Figure 4. The interaction was studied at 3, 5, and 10-day time points. At 3 days, the hECM scaffolds with 4× AuNPs appeared to be the most metabolically active relative to all other groups. At 5 days, the 1× AuNPs hECM appeared to stimulate chondrocyte activity relative to the other groups. The 4× AuNP scaffolds had a reduced viability at 5 days. At 10 days, the samples with curcumin were the most metabolically active. Overall, the samples all appeared to maintain cellular viability over a 10-day time period.

Another cell viability study examined the addition of HA to the hECM as shown in Figure 5. A 3 and 6-day time point were used in this study with chondrocyte cells. At 3 days, the hECM supplemented with HA had the highest cell viability while at 6 days, a significant increase in the hECM supplemented with HA and AuNPs was observed. Each of the samples displayed an increase in cellularity over time, with the Au-HA sample displaying a 1516% change from 3 to 6 days as well as a significant change as compared to the control. The Au group also displayed a 1304% increase from 3 to 6 days while the control and HA groups had a 163% and 136% increase, respectively.

ROS assay

An OxiSelect ROS assay was used to determine ROS production by the L929 fibroblast cells. A 4% curcumin was chosen due to the previous WST-1 study. From Figure 6, on average, the addition of AuNPs appeared to reduce ROS as compared to the control sample. Adding 4% curcumin to the samples with AuNPs resulted in a statistically significant decrease in ROS production (p < 0.05).

We also performed another OxiSelect ROS assay to determine ROS production from equine synovial fibroblast cells. The results are also shown in Figure 6. We utilized 4xAuNPs and compared it to the control. The results demonstrated a significant reduction in ROS (p < 0.05) with the addition of the AuNPs as compared to the control.

The final ROS assay was performed with HCH to determine the ability of hECM to reduce intracellular ROS. The results are shown in Figure 7. The 4× AuNPs, 4% (w/v) curcumin, and 15% HA were all studied. There was a synergistic correlation between ROS production and the addition of AuNPs, HA, and curcumin. Individually, the AuNPs, HA, and curcumin lowered ROS relative to the control samples. Each permutation of the additives also lowered ROS. The lowest ROS was produced when AuNPs, HA, and curcumin were all added to the homogenized ECM.

Apoptosis assay

A HT TiterTACS assay was performed to determine whether the hECM scaffolds will influence human chondrocyte cells apoptosis when stimulated by IL-1B (10 ng/mL). Cells were incubated with IL-1B for 24 h prior to addition of hECM scaffolds. The results are shown in Figure 8. A reduction in apoptosis was observed with the combinatorial addition of AuNP, HA, and curcumin relative to the conjugated and unconjugated homogenized scaffold.

Discussion

Current therapies to treat PTOA include intra-articular injections of pain killers, anti-inflammatory agents, corticosteroid, or HA.²⁵ Unfortunately, there is a rapid breakdown or “washing out” of these materials and a lack of long-term or lasting benefits, which require repeat injections.³⁷ We investigated the use of homogenized, decellularized ECM to determine its chondrocyte biocompatibility and protection ability to serve as a long-lasting therapy.

The utilization of the decellularized ECM as a scaffold material has many advantages. The ECM possesses inherent biochemical cues. It harbors cryptic matrikines (hidden segments of larger proteins), which act as chemoattractants for progenitor cell populations and stimulate the release of growth factors, which protect articular cartilage from degeneration.³⁸ Another benefit is the slowly degrading ECM coordinates a complex biochemical remodeling response over time.^39,40 Additionally, the ECM can be homogenized to allow injectability. It can be conjugated and/or mixed with AuNPs or anti-inflammatory agents, and it can slow down in vivo enzymatic degradation allowing more time for ECM recapitulation.^39–41

The incorporation of AuNPs to the hECM was investigated to determine AuNPs potential as a biocompatible, anti-inflammatory agent. AuNPs were conjugated to the ECM via chemical crosslinkers, and SEM and EDS analysis was utilized to verify the conjugation of AuNPs. Our results demonstrated, as shown in Figures 1 and 2, that the AuNPs conjugated to the ECM structure; EDS analysis confirmed the presence of AuNPs within the scaffolds. Figure 1(a) provides evidence of the hECMs open porous ECM structure prepared through critical point drying while Figures 2(a) and (c) provide information on the natural, hydrated state of the hECM. The amine modified AuNPs created a zero-length, peptide bond with the carboxylic acid groups found in decellularized ECM, allowing conjugation of the AuNPs to the ECM. Our previous studies have demonstrated the ability to conjugate AuNPs, creating a scaffold more resistant to degradation.⁸ It was determined that the AuNPs can slow down in vivo enzymatic degradation due to hindering collagenase binding site on the ECM.^41–46

Extrusion force is an important factor for clinicians when injecting therapeutics. If the force required to inject a therapeutic is too high, it could result in injury to the patient. Table 3 shows the maximum compressive force required to extrude the hECM integrated with various concentrations of HA. A commonly used 22-gauge cannula for orthopedic injections was used in this study.^47,48 All of the HA compositions required less than 1 N of force to extrude. A study by Kim et al.⁴⁹ developed a collagen/HA injectable composite filler which required 10–12 N of force to extrude through a 27-gauge cannula. Another study by Cao et al.⁵⁰ developed an injectable HA composite requiring 2 N of force to extrude through a 26-gauge cannula. In comparison, our hECM injectable required less force 1 N making it well suited for orthopedic injection.

Modulating inflammatory response is a key factor in mitigating the progression of PTOA; we investigated the incorporating of AuNPs, curcumin, and HA in the homogenized ECM as biocompatible, anti-inflammatory agents. Cellular viability studies were first performed in order to examine the effects of AuNPs, curcumin, and HA on the biocompatibility of different cell lines.

Initial work with murine L929 fibroblast cells was conducted to assess overall biocompatibility of the AuNPs and curcumin supplement. A 3-day WST-1 assay was performed, and the results are displayed in Figure 3. The crosslinked hECM served as the control. The results indicated that the addition of AuNPs enhanced the viability of the hECM. This is in agreement with previous published papers on conjugating AuNPs to synthetic and/or biologics.⁵¹ For example, prior work by Smith et al.⁸ provided evidence that a 4× 20 nm AuNP concentration maintained biocompatibility and improved cellular migration. This may be due to the cells being attracted to the higher surface energy of the AuNPs.⁵² While there has been published research about the cytotoxicity of AuNPs,⁵³ we did not observe cytotoxicity. By conjugating AuNPs to the hECM, we are able to prevent cellular uptake and thus mitigating the concern of cell death due to AuNP internalization. The viability was further enhanced with the addition of curcumin. On average, the presence of curcumin demonstrated an increase in viability with the 4% curcumin-AuNPs hECM samples demonstrating the overall highest viability. While some studies have demonstrated cytotoxic effects of curcumin,⁵⁴ we have demonstrated, at the concentration of curcumin utilized and its incorporation into a hECM, increased viability.

Human chondrocyte cells were then utilized to provide a more physiologically relevant examination as to how the hECM would perform in an articular environment. Two WST-1 assays were employed. The first assay examined 1× & 4× AuNP concentrations with and without 4% (w/v) curcumin over a 10-day period as shown in Figure 4. The results using the chondrocytes are in good agreement with the previous study using fibroblasts. In this time study, the viability increased over time with day 10 demonstrating the highest viability of all the tested samples. This indicated that little to no cytotoxic effects were occurring over time. Overall, the hECM with AuNPs and with/without curcumin demonstrated good viability.

In the second WST-1 assay with the HCH, HA was added to the hECM to determine HA effects on chondrocytes at 3 and 6 days as shown in Figure 5. At 3 days, there was no statistical significant difference between the samples. However at day 6, the 4x AuNP + HA homogenized ECM significantly increased cell viability (p < 0.05) over all the other groups and also had a 1516% increase in viability over the 3-day time span. The AuNP group also displayed a similar percent increased viability increasing 1304% between day 3 and day 6. These results indicate that there may be a synergistic, complementary effect between the AuNPs and HA, that the cells are adapting and binding to the AuNPs in this more cell friendly HA environment. Additionally, it is possible that there is a delayed metabolic response from the chondrocyte cells between day 3 and day 6 time points. The results of the WST studies demonstrate the biocompatibility of utilizing AuNPs, curcumin, and HA although concentration and ratio amount may affect the viability of the cells.

The anti-inflammatory effects of the AuNPs, curcumin, and HA were examined using ROS assays. Homogenized extracellular matrix alone was utilized as the control in which we analyzed and compared the response of the cells to the hECM + constituents (AuNP, HA, and/or curcumin). In the first study, we utilized L929 fibroblast cells and examined the effects of ROS production with the AuNPs and curcumin hECM samples as shown in Figure 6. The results demonstrated a relative decrease in ROS production for all the samples; however, the combination of AuNP with curcumin displayed greater reduction. There was a significant difference between the control and the 1xAuNP+curcumin (p < 0.05) samples. Both the curcumin and AuNPs appeared to play a role in the reduction of ROS production. A second ROS study was performed with more cellular relevant cells. Equine synovial fibroblast cells were used to study ROS production in the hECM and in the hECM with AuNPs. A significant reduction in ROS was observed between the control and the 4× AuNPs hECM (p < 0.05) indicating that the AuNPs may be playing a role in the reduction of ROS.

A hallmark of PTOA is high levels of ROS production,^55,56 which is derived from chondrocyte cells after experiencing mechanical stress on the articular cartilage. The production of ROS leads to cellular apoptosis and breakdown of articular cartilage.^57,58 To determine the ROS-mitigating potential of the hECM, an ROS assay was utilized to study the interaction of human chondrocyte cells stimulated with IL-1β. Interleukin-1β was used to stimulate a simulated osteoarthritic state by the chondrocyte cells 24 h prior to adding the hECM.^59–61 Figure 7 demonstrates a decrease in ROS for all the sample groups. Interestingly, the hECM sample combinations of AuNP + HA and the combination of AuNP + curcumin both further reduced ROS in comparison to the individual component of just AuNP, curcumin, or HA. The supplementation of AuNP, curcumin, and HA together demonstrated the highest reduction of ROS on average. There appears to be synergetic, complementary effect with these supplements when taken in combination resulting in the incremental decreases in ROS production. However, optimizing of the amounts of each supplement needs to be performed in order to achieve the optimal effects.

This study demonstrated the anti-inflammatory effect of AuNPs conjugated to hECM. The use of gold and specifically AuNPs has been used as anti-inflammatory agents for years.^62,63 AuNPs are zerovalent, have high surface reactivity, and are resistant to oxidation. The therapeutic effect of AuNPs is believed to be at least partially facilitated by their anti-oxidative nature.^52,63 Specifically, it has been determined that AuNPs act as anti-oxidative agents by inhibiting the formation of reactive oxygen species (ROS) and scavenging free radicals, which lowers oxidative stress levels.⁵²

This study also demonstrated that the presence of curcumin and HA will reduce ROS production. While curcumin is a known suppressor of inflammation through many mechanisms, such as its ability to block NF-κB activation, the utilization of curcumin has been hindered due to low solubility, rapid metabolism, and poor bioavailability.⁶⁴ It is possible that incorporating curcumin into the homogenized ECM as a carrying system, the advantageous properties of curcumin can be preserved. While HA is a natural lubricating component of articular joints, the addition of HA into the hECM also demonstrated reduced ROS production. This work correlates with previous researchers who concluded that HA might reduce ROS through the Nrf2 regulation by activating Akt.⁶⁵

The apoptosis assay was performed in order to examine the chondro-protective ability of the hECM in preventing programmed cell death of the chondrocytes; the results are shown in Figure 8. IL-1B was incubated with chondrocyte cells 24 h prior to addition of the hECM to simulate an osteoarthritic chondrocyte cell environment. A reduction in chondrocytic apoptosis by the AuNP+Curcumin+HA-hECM can be observed. The unsupplemented hECM also appeared to have some chondroprotective ability in comparison to the positive control. The ECM’s inherent biochemical cues most likely aided in protecting cells from programmed cell death. Examining the results of the ROS assay and apoptosis assay using the chondrocyte cells, there is evidence that by introducing the hECM supplemented with AuNPs, curcumin, and HA resulted in the ability to reduce intracellular ROS and also prevent chondrocyte cells from programmed cell death. These two factors are necessary in mitigating PTOA progression. By mitigating cellular apoptosis, chondrocyte cells have the ability to survive the catabolic PTOA environment. By reducing intracellular ROS, the chondrocyte cells have the opportunity to return to their homeostatic regulation mechanisms, which may possibly regulate and maintain articular cartilage.

In summary, we have demonstrated that an injectable hECM supplemented with AuNPs, curcumin, and HA may have applications in mitigating the progression of PTOA. Conjugation of AuNPs to the hECM was successful with further validation of the material to be injected. In vitro analysis was validated using cell viability, ROS, and apoptosis studies with murine fibroblast, equine synovial fibroblast, and human chondrocyte cells. Homogenized extracellular matrix supplement with AuNPs, curcumin, and HA have the best potential of being utilized for PTOA application with the ROS mitigating and chondro-protection results.

Footnotes

Acknowledgments

The authors would like to acknowledge funding from the University of Missouri Food for the 21st Century Program.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study is supported by University of Missouri Food for the 21st Century Program.

ORCID iD

Sheila A Gran

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