GENE ACTIVATED REINFORCED SCAFFOLDS FOR SOX9 DELIVERY TO ENHANCE REPAIR OF LARGE LOAD BEARING ARTICULAR CARTILAGE DEFECTS

Articular cartilage (AC) has a poor capacity to repair once damaged, with progressive degeneration often leading to osteoarthritis (OA). While biomaterials fabricated with extra cellular matrix (ECM) native to the AC have shown promise for repair of focal AC defects, several challenges must be overcome for the repair of larger load bearing defects due to poor scaffold mechanical properties and a lack of chondrogenic potential in diseased cells. Here, we develop a method to improve such biomaterials by incorporating a bioabsorbable 3D printed reinforcing framework and the delivery of pro-chondrogenic genes to infiltrating stem cells to enhance chondrogenesis and produce hyaline tissue that is more indicative of healthy AC. A bioabsorbable polycaprolactone (PCL) 3D printed framework was surface treated to improve its hydrophilicity and used to reinforce a collagen hyaluronic acid (CHyA) matrix. The mechanically reinforced scaffolds were then gene-activated (GA) with the chondrogenic transcription factor SOX9 which was complexed with non-viral nanoparticles (NPs) generated using the glycosaminoglycan-binding enhanced transduction (GET) system, before being seeded with human mesenchymal stromal cells (hMSC). After 28 days culture in chondrogenic media, hMSCs on the GA-scaffolds deposited an ECM more indicative of healthy hyaline cartilage compared to the gene free control. SOX9 mRNA expression on the GA scaffold was 2-orders of magnitude higher than on the control, with downstream chondrogenic targets of SOX9 ( COL2 α 1 , ACAN ) also expressing significantly higher mRNA levels. Expression of pro-chondrogenic ECM proteins such as COL2, were 17.5 times greater ( p = 0.0018) on the GA scaffold which also resulted in enhanced production and spatial distribution of sulphated glycosaminoglycans (sGAG), which are critical to the function of healthy AC. In summary, these findings provide evidence that functionalization of a 3D printed biomimetic pro-chondrogenic scaffold with SOX9 NPs enhances the quality of ECM produced by human stem cells on such mechanically reinforced scaffolds.


Introduction
Articular cartilage (AC) has a poor capacity to repair once damaged, with progressive degeneration often leading to osteoarthritis (OA) and the need for a total joint replacement (Hodgkinson et al., 2022; Medvedeva et al., 2018; Levingstone et al., 2016).Globally, over 527 million individuals suffer from OA, making it the most prevalent disability amongst adults in the United States (US), costing the US healthcare system $45 Billion in 2015 (Long et al., 2022; Scheuing et al., 2023; Li et al., 2016; Zhao et al., 2019).Concerningly, OA prevalence in younger individ-uals is expected to increase in the coming decades, caused in part by increasing obesity rates (Leskinen et al., 2012).Traditionally, artificial total joint implants have been used as a terminal treatment for OA.However, while successful in the short-term, these implants are costly and prone to loosening and failure over time (Rolfson et al., 2009; Deirmengian & Lonner, 2008; Mihalko et al., 2020).Tissue engineering and regenerative medicine offers an alternative approach by focusing on promoting cartilage regeneration prior to severe tissue degeneration and loss-of-function (Billings et al., 1990; Niemeyer & Angele et al., 2022).

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European Cells and Materials Vol.47 2024 (pages 91-108) DOI: 10.22203/eCM.v047a07Recent advancements in biomaterials for AC defect repair have shown success improving cartilage regeneration in focal AC defects (Levingstone et al., 2016; Niemeyer & Angele, 2021).However, biomaterials design for the effective repair of larger AC defects or defects in load bearing regions of cartilage remains a significant challenge as the most successful biomaterials are typically fabricated with extra cellular matrix (ECM) native to the AC.These materials are typically limited in terms of mechanical properties when compared to healthy tissue.Therefore, successful repair strategies must be capable of both withstanding the mechanical loads present in articular cartilage while also promoting and controlling cell-mediated tissue regeneration i.e. retaining biological functionality.
One strategy to address this problem involves the reinforcement of regenerative biomaterials to improve their mechanical properties and more accurately mimic the compressive modulus of healthy AC (0.5-2.0 MPa) (Kabir et al., 2021; Schipani et al., 2020; Visser et al., 2015; Hutmacher et al., 2001).Recently, our lab developed a freezedried microporous matrix for AC repair with a proven track record of promoting chondrogenic differentiation and improving cartilage defect repair (Intini et al., 2022; Levingstone et al., 2016), which is comprised of type-I collagen, type-II collagen, and hyaluronic acid (CHyA) that promotes production of type II collagen and sulphated glycosaminoglycans (sGAG) but no significant increase in type X collagen (Levingstone et al., 2016; Intini et al., 2022; Matsiko et al., 2012; Intini et al., 2022).To adapt this technology for large defect repair, this CHyA matrix was recently mechanically reinforced, through integration of a 3D printed bioabsorbable lattice comprised of polycaprolactone (PCL) (Joyce et al., 2023).This reinforcement increased the compressive modulus of the CHyA scaffold to within the range of native cartilage, while also serving as a secure attachment point to the subchondral bone to ensure adequate cellular and tissue integration when paired with a microfracture surgical technique (Joyce et al., 2023).This approach can feasibly shield biomaterial-resident cells from excessive compressive forces during loading and reduce mechanically-driven increases in inflammation and catabolic enzyme production (Hodgkinson et al., 2022; Selig et al., 2020).Importantly, this approach allows integration of this mechanical reinforcement with softer, regenerative biomaterials that support chondrogenic differentiation and production of articular cartilage-like ECM (Joyce et al., 2023; Lotz et al., 2021; Sheehy et al., 2019).Initial studies have demonstrated that this reinforcing structure can be designed to have a positive impact on chondrogenesis, even in static culture conditions, significantly increasing sulphated glycosaminoglycans (sGAG) deposition (Critchley et al., 2020).However, it is expected that effective treatment of larger area AC defects will require additional stimulation to ensure adequate hyaline tissue formation and to ensure invading cells adopt the correct phe-notype.
To promote AC repair, recombinant growth factors (rGFs) have been typically used to enhance the regenerative potential of biomaterials (Chen et al., 2020; Holland et al., 2005).Unfortunately, the short molecular half-life of rGFs makes delivering an effective and sustained localized dose in vivo difficult.This means that often supraphysiological doses or repeated delivery of rGFs are required, which can limit spatial control and increasing the risk of off-target effects, adverse outcomes and costs (Qi et al., 2019; Lee et al., 2011).By contrast, delivery of pro-chondrogenic gene therapies on chondro-supportive biomaterials have been shown to locally deliver effective and sustained doses, with positive physiological benefits being observed in vitro and in vivo when treating focal AC defects (Raftery et al., 2019; Venkatesan et al., 2018; Venkatesan et al., 2022; Carballo-Pedrares et al., 2022; Raftery et al., 2020).These gene therapies are typically delivered using viral vectors, although our lab has put particular focus on non-viral scaffold-based gene delivery for indications including bone, nerve, skin as well as cartilage (Raftery et al., 2019; Walsh et al., 2021; Laiva et al., 2021; Walsh et al., 2019; Laiva et al., 2021).The aim of this study was to gene activate our previously developed mechanically reinforced biomaterial (PCL-CHyA) (Joyce et al., 2023) with a proven history of supporting chondrogenesis to further enhance production of hyalinelike ECM for the treatment of large area AC defects including in tissues suffering from compromised biological functionality.
Our focus with this gene activated scaffold platform is ultimately to target resident stem cells in the bone marrow beneath the surface of the articular joint.The surgical procedure of microfracture is used commonly clinically to release human mesenchymal stromal cells (hMSC) from the bone marrow to attempt to repair AC defects.However, the resultant tissue formed is predominantly inferior fibrocartilage which is rich in type-I collagen whereas the collagen composition in healthy AC tissue is ~95 % type-II collagen (Sophia et al., 2009; Cohen et al., 1998).We thus propose to use gene delivery of pro-chondrogenic factors to enhance the ability of these MSCs to effectively repair large defects.The SRY-box transcription factor 9 (SOX9) was identified and selected as the target gene based on its well understood and documented role as a master chondrogenic transcription regulator (Rey-Rico et al., 2018; Jo et al., 2014; Haudenschild et al., 2010; Lefebvre et al., 2019).Therefore, we hypothesized that gene activating the mechanically reinforced pro-chondrogenic PCL-CHyA scaffolds would further enhance chondrogenic differentiation of infiltrating hMSCs, leading to higher quality ECM deposition more indicative of healthy articular cartilage ECM, for the purported treatment of larger AC defects.
The specific objective of this study was thus to complex pSOX9 with non-viral nanoparticles (NPs) and deliver them on a mechanically reinforced PCL-CHyA scaf-European Cells and Materials Vol.47 2024 (pages 91-108) DOI: 10.22203/eCM.v047a07fold.Gene delivery was mediated by the use of the Glycosaminoglycan-binding enhanced transduction (GET) system which has previously been successfully exploited in a number of applications (Intini et al., 2023; Eltaher et al., 2022; Power et al., 2022; Blokpoel et al., 2021; Blokpoel et al., 2020; Abu et al., 2020; Jalal & Dixon, 2020; Ritchie et al., 2020).Validation of the SOX9 activated scaffolds were conducted by assessing their ability to promote the chondrogenic differentiation of hMSCs compared to gene free control scaffolds.Expression of pro-chondrogenic, and hypertrophic genes, along with secreted ECM components were assessed to determine the effect of SOX9 activated scaffolds on chondrogenic differentiation.The developed approach has great potential to be used as an off-the-shelf solution for the treatment of large area AC defects which have very limited treatment options currently available.

Biofabrication of Mechanically Reinforced Biomimetic Scaffolds
A collagen and hyaluronic acid (CHyA) slurry was created by blending 0.9g of bovine type I collagen (Collagen Matrix, Paramus, NJ, USA) (0.25 % w/v) and 0.9 g of porcine type II collagen (Symatese, Chaponost, France) (0.25 % w/v) in 300 mL of 0.5 M acetic acid.Then 0.18 g of hyaluronic acid (1.50-1.80MDa) (Contipro, Dolní Dobrouč, Czech Republic) (0.05 % w/v) was added dropwise as previously described by Matsiko et al. (2012).The CHyA slurry was combined with 3D printed PCL scaffolds as described (Joyce et al., 2023).Briefly, PCL (Polysciences, Hirschberg an der Bergstrasse, Germany) scaffolds (2 mm height × 10 mm diameter) were 3D printed with an Allevi II (Allevi, Philadelphia, PA, USA) with a continuous non-intersecting gyroid infill pattern.PCL scaffolds were surface treated in 3 M NaOH for 48 hours, to cleave ester bonds on the surface of the PCL making it more hydrophilic, while also micro-etching the surface of the PCL to increase its roughness on the microscopic scale.Surface treated gyroid scaffolds were rinsed three times with diH 2 O to remove excess NaOH.PCL scaffolds were placed in a cylindrical steal mold with CHyA slurry being pipetted into the mold to occupy the remaining space, before they were degassed together.The complexed PCL-CHyA then underwent a 48-hour freeze drying cycle to -20 °C as previously described by Matsiko et al. (2012), (Joyce et al., 2023; Matsiko et al., 2015; Haugh et al., 2011).After freeze-drying was complete, PCL-CHyA scaffolds were crosslinked for two hours at pH 5.0 with 1-Ethyl-3-(3(dimethlaminopropyl)-carbodimide (EDAC) (Sigma-Aldrich, St. Louis, MO, USA) and N-Hydroxysuccinimide (NHS) (Sigma-Aldrich, St. Louis, MO, USA) in a 5:2 molar ratio (EDAC:NHS) using 70 % EtOH as a solvent.Scaffolds were then rinsed three times with sterile PBS prior to use.

Nanoparticle Complexation and Optimization
SOX9 nanoparticle (NP) complexation was performed as previously described (Raftery et al., 2019).In short, 3 different charge ratios (CR: 6, 9, 12) were complexed by varying the relative mass of a cell penetrating peptide (GET) to the mass of the pDNA cargo within OptiMEM (30 µL) (Gibco, Loughborough, UK).We have previously used the GET peptide for gene delivery in a number of indications (Raftery et al., 2019; Intini et al., 2023; Eltaher et al., 2022; Power et al., 2022; Markides et al., 2019).To compare transfection efficiencies of the SOX9 GET NPs to a commercially available lipid nanoparticle (LNP) vector system, Lipofectamine TM 3000 (L3000015, ThermoFisher Scientific, Carlsbad, CA, USA) was utilized based on its documented use and availability commercially (Khaitov et al., 2021; Park et al., 2011).Equal amounts of pDNA were delivered with LNP as with GET and complexed according to the manufacturer's protocol (Thermo Fisher Scientific Inc, 2016).pDNA quantity between 2D, and 3D experiments were scaled relative to cell number, with ~4 picograms of pDNA delivered per hMSC.

Gene Activation of Reinforced Scaffolds with SOX9 Nanoparticles
Prior to cellular seeding, sterile cylindrical PCL-CHyA scaffolds (2 mm × 10 mm) had 1 µg of pDNA with complexed vectors (GET, LNP) suspended in OptiMEM, pipetted onto the superficial cylindrical face, before the microporous scaffold was flipped and the opposing face had an additional 1 µg of pDNA pipetted onto it, making a total of 2 µg pDNA per gene activated scaffold.The LNP control was pipetted onto the scaffold similar to the SOX9 NPs.However, 4 hours after seeding cells on the LNP scaffolds, a media change was performed with growth media (GM) as previously described by Khaitov et al. (2021) to decrease the cytotoxicity of Lipofectamine 3000 (Thermo Fisher Scientific Inc, 2016).Whereas cells seeded on SOX9 activated scaffolds remained in the initial GM for 24 hours before receiving a media change to CCM.

Maximizing NP Transfection Efficiency for hMSCs on 3D Reinforced Scaffolds
To maximize transfection efficiency of hMSC within the pro-chondrogenic scaffolds, 3 different charge ratios (CR) of 6, 9, and 12 were complexed with a reporter plasmid (pGaussia Luciferase) by varying the mass of GET NPs to pDNA.Media samples were collected at 24, 72, and 168 hours and analyzed with a Luciferase Flash Assay (Thermo Fisher Scientific, Bridgewater, NJ, USA), to determine transfection efficiency non-destructively.

Validation and Spatial Characterization of SOX9 Transgene Expression with Immunocytochemistry
Immunocytochemistry (ICC) was performed to confirm that pSOX9 transfections led to an increase in SOX9 protein and determine if SOX9 was successfully shuttled into the nucleus.Therefore, 7 days after transfection in 2D culture ICC analysis was performed with antibodies specific for SOX9 (1:250, ab182579, Abcam, UK), using a secondary antibody conjugated with Alexa Fluor 568 (1:200, ab175473, UK).Samples were counterstained with DAPI at a 1:500 dilution before being imaged with a Leica DFC520C camera (Leica Microsystems, Wetzlar, Germany) on a microscope (AE31E, Motic, Germany).Confirmation of transfection and spatial localization was performed visually by overlapping images of DAPI nuclear staining with SOX9 antibody expression with ImageJ software version 1.53K (LOCI, University of Wisconsin, Madison, WI, USA).The percentage of transfected cells was calculated by counting SOX9 positive cells relative to total DAPI stained cells.

Analysis of Pro-Chondrogenic Protein Expression
Western blot (WB) protein analysis was performed to compare relative protein expression between hMSCs on SOX9 activated scaffolds and their gene free control.For SDS-PAGE, 15 µg of denatured total protein samples from individual biological repeats with 4× Laemmli sample buffer (#1610747, BioRad, Watford, UK) were loaded and ran in Mini-PROTEAN TGX electrophoresis gels (#4561094, BioRad, Watford, UK) at 150 volts for 50 minutes.Proteins within the polyacrylamide gels were then transferred to 0.2 µm PVDF membranes with a Trans-Blot Turbo Transfer Pack (#1704156, BioRad, Watford, UK).
Membranes were blocked (5 % (w/v) powdered milk in tris-buffered saline with 0.1 % tween-20 TBST) and incubated overnight at 4 °C with a primary rabbit antibody specific to human SOX9 (ab182579), or COL2A1 (ab188570).Membranes were washed 3 times with TBST, before membranes were incubated in a secondary antibody (goat antirabbit IgG-HRP (ab205718)) for one hour at room temperature.After washing three times in TBST, membranes were treated with 1mL of enhanced chemiluminescence (ECL) substrate (BioRad, Watford, UK) and imaged on a GE Amersham 600 chemiluminescent imager.Membranes were stripped (15 g Glycine, 1 g SDS, 10 mL Tween-20, 1 L diH 2 O) at a pH of 2.2, before blocking and incubation with GAPDH (ab9485) antibodies at a 1:5000 dilution.As previously described, membranes were then washed, incubated with HRP conjugated secondary antibodies (goat anti-rabbit IgG-HRP) (ab205718, 1:5000) and imaged in the presence of ECL.Relative protein expression was quantified using ImageJ software to determine signal band intensity relative to the expression of the correlative GAPDH band from each scaffold.

Characterization of cellular health through metabolic activity
An alamarBlue Assay (ThermoFisher Scientific, Eugene, OR, USA) was used as a non-destructive indicator of cellular metabolic viability without terminating the experiment.Cell seeded PCL-CHyA scaffolds were incubated with 10 % AlamarBlue in CCM for 2 hours.Media samples were then measured with a 560 nm/590 nm excitation and emission wavelength respectively to determine relative metabolic activity of cells.Due to the large size, and opaque nature of the 3D microporous PCL-CHyA scaffold, live/dead assays are not an accurate representation of cel-European Cells and Materials Vol.47 2024 (pages 91-108) DOI: 10.22203/eCM.v047a07lular viability, as they are in suspension, or 2D cell culture characterizations (MacCraith et al., 2022).

DNA and sGAG Quantification
Sulphated glycosaminoglycans (sGAG) and DNA quantification were both performed by first lysing cell seeded scaffolds with consecutive freeze-thaw cycles prior to digestion with 0.01 % papain (Sigma-Aldrich, Wicklow, Ireland) overnight at 65 °C.A Blyscan sGAG assay (Biocolor, Carrickfergus, UK) (Matsiko et al., 2015; Manual, 2012), and a Quant-iT PicoGreen dsDNA assay (Invitrogen, Waltham, MA, USA) (ThermoFisher, 2008) were used following their respective manufacturer protocols.In brief, sGAGs were quantified through binding of Blyscan dye and absorbance measured at a wavelength of 656 nm.sGAG concentration was determined by comparison to a standard curve.Similarly, DNA was quantified by intercalating ds-DNA with Picogreen, and measuring the fluorescence at a wavelength of 538 nm.dsDNA concentration was also determined by comparison to a standard curve.

Histological Analysis of ECM Deposition
After 28 days in cell culture, scaffolds were rinsed with PBS and fixed in 4 % paraformaldehyde for histological staining.Scaffolds were dehydrated in an ethanol gradient before soaking in Xylene (#534056, Sigma-Aldrich, Overijse, Belgium) to dissolve the reinforcing PCL structure prior to paraffin wax (8002-74-2, Epredia, Kalamazo, MI, USA) embedding.Scaffolds were serially crosssectioned (10 µm thick) in the axial plane from the top cylindrical face to the bottom, with a microtome (RM2255, Leica Microsystems, Wetzlar, Germany).Histological sections were deparaffinized and rehydrated through an ethanol gradient before staining with 2 % Alcian Blue (pH = 1) (A5268, Sigma-Aldrich, USA) and a nuclear-red counter stain (#1.00121,Merck, Darmstadt, Germany).After staining, samples were imaged with a Nikon Eclipse 90i microscope (Nikon Co, Tokyo, Japan) and Nikon DS RiL camera (Nikon Co, Tokyo, Japan).Entire cross-sectional slices of scaffolds were consecutively imaged at 10× magnification to create high resolution mosaic images.For transparency, full-layer histological slices are shown at incremental depths, in addition to magnified regions of interest, to give the fairest representation of results.
Additionally, immunohistochemical (IHC) analyses were performed on histological sections to characterize the extracellular matrix deposition spatially.Sections from the upper superficial region (0-500 µm depth) were incubated overnight at 4 °C with a mouse primary antibody for COL2 (SC52658, Santa Cruz, CA, USA) at a 1:100.A secondary antibody specific to mouse IgG conjugated with HRP (ab6728, Abcam, Cambridge, UK) in a 1:500 dilution was incubated for 1 hour.Samples were treated with Avidin Peroxidase in blocking buffer (45 min).Then treated with a DAB substrate peroxidase kit (SK-4100, Vector Labora-tories, Newark, CA, USA) for 10 minutes.Sections were mounted with coverslips and imaged with a Nikon DS RiL camera as previously.

Statistical Analysis
3D cell culture experiments were repeated with 3 separate donor populations of hMSCs.Each donor had its own biological repeats (n = 3).Data from the 9 scaffolds from each treatment group were weighted equally, n = 9.Graphical data points are color coded to denote different donor populations ( Donor 1, Donor 2, Donor 3).Significant statistical differences between treatment groups were calculated using Prism GraphPad software version 9.3.1 (Prism, San Diego, CA, USA).One-way analysis of variance (ANOVA) with a Tukey post-hoc test for multiple comparisons between all groups, with a 95 % confidence interval (CI) was utilized when appropriate.Alternatively at times as indicated in figure legends, ANOVA with Fisher's LSD test were utilized when appropriate.When comparing only two separate treatments, two tailed T tests were performed.All figures have p values expressed as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.00001.

NP Charge Ratio was Optimized for hMSC Transfection in 3D Pro-Chondrogenic Reinforced Scaffolds
To determine the optimal charge ratio of GET NP to pDNA to transfect hMSCs within 3D scaffolds (2 mm × 10 mm), a reporter plasmid (Gaussia Luciferase) was complexed with the delivery peptide at three different charge ratios (6, 9, 12) (Fig. 1a).No significant difference in luciferase expression was observed between the 3 CR treatment groups and the gene free control at the 24-hour timepoint.However, there was a significant increase in secreted luciferase 3 days after transfection in all three treatment groups compared to the gene free control.Only two of the three treatment groups (CR6, CR9) sustained significant production of luciferase expression 7 days post transfection.Luciferase expression in the CR12 group was not significantly different from the gene free control at D7 (p = 0.4081).Since CR9 presented the highest luciferase expression in 2/3 timepoints, this CR was selected for all future transfections on gene activated scaffolds.

Successful SOX9 Upregulation Lead to Increased Nuclear SOX9
Successful transfection of hMSCs with SOX9 NPs in 2D culture was confirmed through ICC at 7 days posttransfection, with a detectable upregulation of the SOX9 protein observed in comparison to gene free controls (Fig. 1b).Importantly ICC confirmed with DAPI counterstaining that detectable SOX9 protein was spatially located within the nucleus, which is required for it to perform as a pro-chondrogenic transcriptional regulator.While basal levels were observed in the gene free sample with 6 % of DAPI + cells expressing detectable levels of SOX9.Expression was enhanced with treatment of SOX9 NPs, and LPN transfection groups with their respective 57 % and 35 % of DAPI + cells expressing detectable levels of SOX9 expression.

hMSCs on SOX9 Activated Scaffolds Maintain Normal Metabolic Activity and DNA Synthesis
Metabolic activity of hMSCs on reinforced scaffolds were measured as a non-invasive indication of cellular health.There was no significant reduction in metabolic activity in hMSCs cultured on the SOX9 activated scaffold compared to the gene free scaffold control after 3 (Fig. 1c), or 26 days (Fig. 1d).Conversely, hMSCs cultured on LNP control scaffolds showed a significant reduction in their metabolic activity compared to the control after 3 days (p = 0.0407) (Fig. 1c).After 26 days in culture the LNP control group was able to recover slightly with no significant change in metabolic activity from the gene free control (Fig. 1d).However, DNA quantification revealed significantly lower levels of DNA present at the experiment end point (Day 28) in LNP scaffold groups than both the gene free and SOX9 NP treatment groups (p = 0.0008, p = 0.0013, respectively).There was no significant difference in quantified DNA between the SOX9 activated and the gene free control (p = 0.9807) (Fig. 1e).

Gene Activated Scaffolds Upregulated SOX9 mRNA Expression at Day 7
Reinforced scaffolds were gene activated with SOX9 NPs to assess their effect on chondrogenic differentiation of hMSCs in culture.After seven days in culture, qRT-PCR was utilized to compare gene expression of SOX9, COL2a1, and ACAN between cells on SOX9 activated and gene free scaffolds (Fig. 2).Individually all three hMSC donor populations had significant increases in the SOX9 mRNA (n = 3, p = 0.0248, p = 0.0467, p = 0.0007 respectively) expression after 7 days, when cultured on SOX9 activated scaffolds in comparison to gene free controls.Although, when the 3 datasets were merged, the total variance between the 3 donor groups meant that a statistically significant increase in SOX9 mRNA was not observed (p = 0.0631).Upregulation of COL2α1 mRNA on SOX9 activated scaffolds was significant (n = 9, p = 0.017), whereas the increase in ACAN expression was not significantly different (n = 9, p = 0.1080) from the gene free control.

Gene Activated Scaffolds Led to Elevated SOX9 Protein Expression in hMSCs
Seven days after hMSCs were seeded on the SOX9 activated scaffolds or gene free control, SOX9 protein expression was assessed by western blot.Cells on the SOX9 activated scaffolds had greater expression of SOX9 compared to cells on the gene free control (Fig. 3a).This was con-firmed by quantifying the relative intensity of SOX9 protein band normalized to their GAPDH protein expression (Fig. 3b).

hMSCs Cultured on SOX9 Activated Scaffolds Upregulated Pro-Chondrogenic Genes
After 28 days in culture the ability of SOX9 activated scaffolds to induce hMSC chondrogenic differentiation was quantifiably evident.qRT-PCR indicated that both SOX9 NPs and LPN were effective at raising SOX9 mRNA expression by two orders of magnitude over the gene free control (Fig. 4a).While the increase was insignificant, this is likely caused by the variance (σ) in how individual donor populations responded to the treatment conditions (σ, gene free = 0.429, SOX9 activated = 151,379, LNP = 476,189).When looking at downstream pro-chondrogenic targets that SOX9 directly regulates, such as type II collagen (COL2α1) (Fig. 4b) or aggrecan (ACAN) (Fig. 4c), both genes were significantly upregulated on the SOX9 activated scaffold compared to gene free control, even with donor variation (σ = 3262.91).In contrast, the LNP control showed no increase in mRNA expression of downstream SOX9 targets (COL2α1, ACAN), which was a somewhat surprising finding since both SOX9 NP activated and LNP seemed to have similar success in upregulating SOX9 mRNA.
Expression of the other SOX-trio pro-chondrogenic transcriptional factors SOX5 (Fig. 4d) and SOX6 (Fig. 4e) was also analyzed.Only hMSCs cultured on SOX9 activated scaffolds demonstrated a significant increase in both SOX5 and SOX6 mRNA.Expression levels of hypertrophic genes COLX (Fig. 4f), and RUNX2 (Fig. 4g) were then analyzed.While there was a slight increase in the mean expression of COLX from hMSCs on the SOX9 activated compared to the gene free control, the increase was not significant (p > 0.05).Similarly, hMSC on SOX9 activated scaffolds showed no significant increase in RUNX2 versus the gene free control.Interestingly, though not statistically significant (p = 0.2407), hMSC on SOX9 activated scaffolds had a 27 % reduction in mean RUNX2 mRNA expression compared to the gene free control.

SOX9 upregulation enhanced COL2 protein production
Along with qRT-PCR, western blot analysis 28 days after hMSCs were seeded on SOX9 activated, gene free, or LNP controls confirmed that SOX9 NP activation resulted in physiologically relevant increase of essential hyaline cartilage ECM protein COL2 (Kannu et al., 2012).hMSCs from all 3 donors exhibited a significant increase in COL2 protein deposition on SOX9 activated scaffolds when compared with their gene free control (Fig. 5a,b).Interestingly, hMSC from one donor (Donor 1) on the LNP scaffold produced a significant increase in COL2 over its gene free control (n = 3, p = 0.0425).However, this trend was not observed with hMSC from the other 2 donors, with no significant change in COL2 expression in LNP samples observed  when aggregating the data from the 3 donors (n = 9, p = 0.97).

SOX9 Activation Enhanced Hyaline Matrix Deposition by hMSC on Reinforced Scaffolds
After 28 days in vitro, sGAG deposited from hMSC seeded on SOX9 activated scaffolds, LNP and gene free controls was quantified to assess chondrogenic differentiation.hMSCs on the SOX9 activated scaffold produced a significantly greater quantity of sGAG compared with the gene free control (p = 0.0427) (Fig. 6a), and LNP control (p = 0.0002).When normalized to DNA content per scaffold, the SOX9 NPs treatment maintained a significant increase in sGAG/DNA (p = 0.0453) over the gene free con- trol (Fig. 6b).To reduce variance stemming from different basal levels of sGAG production amongst donor groups.Total sGAG, and sGAG/DNA were further normalized as a fold change of mean sGAG per donor specific gene free control scaffolds to better elucidate how treatment conditions were affecting cellular sGAG production.Under these conditions, there was a significant increase in the relative sGAG/scaffold production by cells in the SOX9 activated scaffolds compared with the gene free control (p = 0.007) (Fig. 6c).The LNP control produced significantly less sGAG compared to the gene free control (p = 0.0015).Similarly, there was a significant increase in donor normalized sGAG/DNA from cells on the SOX9 activated scaffolds compared with the gene free control (p = 0.0051), or LNP control (p = 0.0012) (Fig. 6d).

Improved Distribution of sGAG on Gene Activated Scaffolds
After 28 days culture, histological assessment of the distribution of sGAG produced by hMSCs cultured on the SOX9 activated scaffold confirmed production of an ECM more indicative of healthy hyaline cartilage when compared to gene free control.Serial cross-sections with 4 representative images from the upper (1500-2000 µm), upper central (1000-1500 µm), lower central (500-1000 µm), and bottom (0-500 µm) regions of the scaffolds show greater sGAG distribution throughout all stratifications of the reinforced SOX9 activated scaffolds (Fig. 7a).A magnified analysis of the upper-central region indicated higher cell density on SOX9 activated scaffold, with more circular nuclei and larger cell volume, which is more indicative of a chondrocyte morphology, compared to the gene free control (Fig. 7b).

COL2 Protein Deposition and Distribution was Improved in Gene Activated Scaffolds
Immunohistochemistry confirmed and corroborated qRT-PCR and WB data indicating greater COL2 protein deposition in SOX9 activated scaffolds.Representative whole slice layers (10 µm thick) were imaged from all three donor groups (Fig. 8a,b).IHC staining indicated enhanced COL2 deposition in SOX9 activated scaffolds when compared with respective gene free controls.With magnified regions of interest (Fig. 8c,d) indicating that COL2 deposition in SOX9 activated scaffolds occupied the void space within the existing scaffold microporous architecture, whereas COL2 deposition on control scaffolds appeared confined to the surface of the microporous architecture.These findings are well aligned with the qRT-PCR and WB findings and further provide evidence that gene activation of a pro-chondrogenic mechanically reinforced PCL-CHyA scaffolds with SOX9 NPs enhanced chondrogenic differentiation of hMSCs, which in turn enhanced the production of highly specialized ECM markers indicative of healthy hyaline tissue.

Discussion
The objective of this study was to gene activate a mechanically reinforced biomaterial with a proven history of supporting chondrogenesis to further enhance production of hyaline-like ECM for the eventual treatment of large area AC defects including in tissues suffering from compromised biological functionality.The increasing prevalence of OA amongst younger populations, particularly following traumatic injury (post-traumatic OA) has motivated increased demand for innovative tissue engineering solutions to pre-emptively repair AC defects before the onset of OA (Pamilo et al., 2022; Inacio et al., 2017).To accomplish this goal, several challenges were addressed, including the engineering of a regenerative scaffold capable of both resisting the mechanical forces present in the joint and promoting the regeneration of articular-like cartilage through gene activation with SOX9 nanoparticles.We demonstrated that, following optimization of the GET NP and scaffold composition, that incorporating SOX9 NPs into the reinforced scaffold effectively transfected and promoted chondrogenic gene and protein expression in infiltrating hMSCs as they migrated into the scaffold.Their enhanced chondrogenic phenotype resulted in significantly enhanced production of specialized ECM components characteristic of articular cartilage, such as COL2 and sGAG, while not upregulating the expression of hypertrophic or fibrocartilage markers such as COLX and RUNX2.We propose this research has substantial impact as it expands the indication of use of a promising natural polymer-based pro-chondrogenic biomaterial to potentially allow effective treatment of large load bearing AC defects which are commonly seen clinically.
Delivery of SOX9 NPs incorporated into the reinforced scaffold was highly effective in this study and resulted in a > 300-fold upregulation of SOX9 mRNA expression in hMSCs when compared to the gene free control.Importantly for translation of this technology into human cartilage, a slowly healing tissue, this expression remained elevated even after 28 days in culture.Upregulation European Cells and Materials Vol.47 2024 (pages 91-108) DOI: 10.22203/eCM.v047a07 of SOX9 also resulted in significant upregulation of downstream transcriptional targets COL2α1 and ACAN, along with other pro-chondrogenic transcription factors SOX5 and SOX6 after 28 days.Both western blot analysis and immunohistological staining indicated superior COL2 protein expression compared to the gene free scaffolds.Similarly greater sGAG production on SOX9 activated scaffolds was quantified and corroborated with Alcian Blue staining.These downstream effects following SOX9 upregulation are significant as they are central to the success of this approach but also are not always observed in previous studies involving upregulation of SOX9 (Carballo-Pedrares et al., 2022; Kupcsik et al., 2010; Kim et al., 2011).Indeed, in this study, LNP controls were observed to increase SOX9 mRNA expression in hMSCs to similar levels observed with GET NPs but failed to demonstrate any benefit on downstream upregulation of chondrogenic SOX9 targets like COL2α1, and ACAN.Similarly, Carballo-Pedrares et al. (2022) reported a less than 2 fold increase in COL2α1 mRNA after lipofectamine-mediated delivery of SOX9 to hMSCs.Here, hMSCs on the SOX9 activated scaffolds with GET NPs had normal metabolic activity relative to gene free controls, while LNP delivery had a negative impact on the metabolic activity of hMSCs, which likely resulted in decreased chondrogenic gene expression downstream of SOX9 (Fig. 1c-e).Taken together, these results indicate the non-trivial nature of the gene activated reinforced scaffold platform design developed here and the strong potential for the developed SOX9 activated scaffold system for future in vivo translation and utility in repair of large area load bearing chondral defects.
A strength of this study was the design and synergistic integration of mechanically reinforced biomaterials, with pro-chondrogenic gene delivery technologies to create an off-the-shelf gene-activated, reinforced regenerative biomaterial.While previous studies have individually documented the advantage of mechanical reinforcement or gene therapies on cartilage regeneration (Matsiko et al., 2012; Intini et al., 2022; Raftery et al., 2020; Matsiko et al., 2015), to the best of our knowledge there are no published studies that complex reinforced biomaterials with compressive moduli equivalent to healthy cartilage with gene therapies to further promote chondrogenesis (Yang et al., 2020).The promising in vitro results obtained here indicate the combination of mechanically reinforced SOX9 activated scaffolds with a common surgical microfracture technique to release bone marrow derived MSCs in vivo may improve the articular cartilage-like characteristics of regenerated tissue, enhancing a biomaterial approach that has shown excellent efficacy for focal defect repair (Levingstone et al., 2016; Raftery et al., 2019; Levingstone et al., 2016).
The results described in this study were obtained in static and normoxic culture conditions, which have previously been reported to be suboptimal for chondrogenic differentiation of hMSCs (Grodzinsky et al., 2000; Anderson & Johnstone, 2017).We note that previous studies involving pSOX9 delivery have indicated expression of SOX9 transgene also requires mechanically-loaded culture conditions to produce significant increases in sGAG secretion by hMSCs (Kupcsik et al., 2010).In this study, the synergistic effect of our gene delivery system and reinforced regenerative scaffold was sufficient to significantly increase sGAG production, without the need for additional mechanical loading.Furthermore, it is likely that the presence of the reinforced framework in SOX9 activated scaffolds, which increased the compressive modulus to mimic the native range of healthy AC, will enhance cell responses further in an in vivo setting by allowing loading and thus enhancing the mechanoresponsiveness of the cells while shielding them from harmful injurious stresses that might have occurred in a non-reinforced scaffold.The intrinsic properties of the SOX9 activated scaffold resulting from reinforcement could thus impact cell-level anabolic responses within scaffolds through local alterations in ECM stiffness sensing and mechano-transductive signaling (Hodgkinson et al., 2022).Though not investigated directly, the results presented here demonstrating increased sGAG, are in agreement with our previous work comparing sGAG production in reinforced and non-reinforced regenerative scaffolds (Selig et al., 2020; Hall, 2019).
Localization and longevity of gene delivery to articular cartilage remains a challenge for effective regeneration.The non-viral SOX9 activated scaffold developed here resulted in a 100× upregulation in mean SOX9 mRNA expression at 7 days, which was sustained for at least 28 days.Similarly, our results show a significant 5-fold increase in COL2 mRNA expression at day 7 (Fig. 2) which develops to a 60-fold (Fig. 4b) increase at day 28.This system compares favourably to alternative approaches involving functionalization of biomaterials for viral gene delivery (Venkatesan et al., 2020), resulting in greater transgene and downstream chondrogenic gene expression, while providing safety and ease-of-production benefits.These in vitro results provide promising evidence that this approach can locally deliver physiologically relevant concentrations of SOX9 to bone marrow derived MSCs invading SOX9 activated scaffolds in vivo.In support of this, a prochondrogenic biomaterial previously developed by our laboratory, which was functionalized for simultaneous delivery of plasmids for the SOX-Trio factors (pSOX5, pSOX6, pSOX9) resulted in excellent chondrogenic responses that were comparable in vitro and in vivo (Raftery et al., 2020).The results presented in the current paper delivering a single SOX9 plasmid on a mechanically reinforced scaffold produced comparable results in vitro to those reported when previously delivering three chondrogenic plasmids, streamlining this previously successful approach.Additionally, this method could be adapted to knock down or silence gene expression of anti-chondrogenic and proinflammatory pathways by delivering micro-RNA (miR) or www.ecmjournal.orgEuropean Cells and Materials Vol.47 2024 (pages 91-108) DOI: 10.22203/eCM.v047a07small interfering RNA (siRNA) in addition to upregulating SOX9 (Castaño et al., 2023).Furthermore, reinforcement increases the utility of the gene activated scaffold for applications involving load bearing cartilage regions and large defects where biomaterials are prone to delamination from defect sites.Moreover, these findings suggest the PCL reinforcing framework did not interfere with GET NPs ability to effectively transfect infiltrating hMSC compared with non-reinforced CHyA matrices (Joyce et al., 2023; Raftery et al., 2020).With no effective surgical interventions currently available other than total joint replacement surgeries, which are considered a terminal treatment option, mechanically reinforced SOX9 activated scaffolds could fulfill the niche demand for an intermediate surgical intervention to treat large area AC defects.This would potentially increase the quality of life for patients, and potentially mitigating the progression of OA, and its ever-increasing burden on global healthcare systems.
Taken together, these gene activated mechanically reinforced scaffolds show potential as an off-the-shelf biomaterial to repair large area, load bearing chondral defects.Though an in vivo study is required to further validate its potential for successful clinical translation, these findings are promising.Additionally, peripheral areas of research may also benefit from this study, as the techniques utilized here could be tailored for other tissue specific applications.For example, dental or bone tissue engineering applications could benefit from the stiffer reinforcing scaffold described here, while gene activation of periodontal, or osteogenic genes with similar techniques as described here might further enhance healing (Li et al., 2022; Tsutsui, 2020; Cunniffe et al., 2017).While the SOX9 activated scaffold was specifically designed to treat load bearing articular cartilage defects, similar biomaterials could thus be biofabricated to regenerate many different types of tissue.

Conclusion
This study successfully gene activated a mechanically reinforced biomimetic scaffold with a master chondrogenic regulator leading to an enhanced ECM production indicative of healthy articular cartilage.Targeted upregulation of SOX9 was sufficient to promote expression of SOX5 & SOX6 transcription factors-known together as the SOX-Trio chondrogenic factors.Downstream transcriptional targets of SOX9 (COL2, ACAN) were also upregulated leading to higher quality ECM production, more closely representing healthy hyaline cartilage while hypertrophic genes; COLX and RUNX2 were not significantly upregulated.Therefore, gene activation with the GET system to deliver DNA in reinforced biomimetic scaffolds could offer an offthe-shelf solution to repair large, load bearing articular cartilage defects.This novel biomaterial has immense clinical translation potential, offering orthopedic surgeons a new tool to regenerate damaged cartilage tissue rather than replacing it.

Fig. 2 .
Fig. 2. Gene expression 7 days after transfection confirmed increased SOX9 mRNA expression in hMSCs cultured on SOX9 activated scaffolds.Expression of the SOX9 transgene, and its downstream targets COL2α1, and ACAN were analyzed with qRT-PCR from cells on gene free (GF) and SOX9 gene activated (GA) scaffolds.Individual data from 3 donors n = 3, and a merger of all donor data sets equally weighted (n = 9) are displayed.A two-tailed mean value T test determined significance of differences between mean values.(*p < 0.05, ***p < 0.001) ( Donor 1, Donor 2, Donor 3).

Fig. 7 .
Fig. 7. Greater sGAG deposition was observed from hMSCs cultured on the SOX9 activated scaffolds compared to gene free control.Representative cross-section images from upper, upper-central, magnified upper-central (scale = 200 µm), lower-central, and bottom regions of the gene free control (a), and SOX9 activated scaffold (b) stained with Alcian Blue (pH1), and counterstained with nuclear fast red.Scale bar = 3000 µm.