Alex H. P. Chan

Modulating how macrophages sense mechanical cues offers a novel strategy to control fibrosis around implanted biomaterials. We term this approach ‘mechano-immunotherapy’, which involves the desensitization of immune mechanosensory pathways to control the host response. Here, we use RN-1734 (RN), a model small molecule to demonstrate the proof-of-concept that pharmacologically disrupting macrophage mechanosensation can mitigate fibrosis. In vitro, RN reduced calcium influx and pro-inflammatory cytokine secretion in J774. a2 macrophages. These effects were strictly context-dependent with efficacy observed only in macrophages on high-stiffness (10% w/v) GelMA hydrogels, with no significant impact on those in softer (5% w/v) hydrogels. In vivo, RN selectively attenuated fibrotic capsule formation around implanted electrospun scaffolds but not smooth hydrogels. Notably, despite hydrogels releasing ∼6-fold more drug than scaffolds, fibrosis was reduced only in the scaffold group, suggesting that therapeutic efficacy is driven by the inhibition of high mechanosensory input rather than the loaded drug concentration alone. Spatial transcriptomics revealed that macrophages acted as the primary mechanosensors at the tissue-implant interface. Unsupervised global principal component analysis revealed that RN acted predominantly on early day 3 macrophages. The strongest effect was observed in surface-adhered mechanosensitive macrophages, where RN treatment enhanced their M2-like phenotype and promoted their dispersal from clustered aggregates into broader distribution within the scaffold. This redistribution was accompanied by a marked reduction in the recruitment of interstitial macrophages from the surrounding tissue, which were enriched for matrix-forming gene signatures. Together, these findings suggest that pharmacological desensitization of immune mechanosensors may represent a promising, context-specific approach to improve biomaterial integration.

Despite the therapeutic potential of mesenchymal stromal cells (MSC), there is limited understanding of optimal extracellular matrix (ECM) environments to manufacture these cells. We developed tissue chips to study the effects of multi-factorial ECM environments under manufacturable stiffness ranges and multi-component ECM compositions. Manufacturing qualities of cell expansion potential, immunomodulation, and differentiation capacity were examined. The results show stiffness effects, with 900 kPa substrates supporting higher proliferation and osteogenic differentiation, along with anti-inflammatory IL-10 expression, whereas 150 kPa substrates promoted adipogenic differentiation at 150 kPa, suggesting that optimal ECM environments may differ based on manufacturing goals. ECM biochemistries containing fibronectin and laminin further modulated MSC manufacturing qualities across various stiffnesses. Proteomic and transcriptomic analyses revealed unique ECM combinations that induced higher levels of angiogenic and immunomodulatory cytokines, compared to single factor ECMs. These findings demonstrate that optimized ECM environments enhance MSC manufacturing quality.

Skeletal myofibers naturally regenerate after damage; however, impaired muscle function can result in cases when a prominent portion of skeletal muscle mass is lost, for example, following traumatic muscle injury. Volumetric muscle loss can be modeled in mice using a surgical model of muscle ablation to study the pathology of volumetric muscle loss and to test experimental treatments, such as the implantation of acellular scaffolds, which promote de novo myogenesis and angiogenesis. Here we provide step-by-step instructions to perform full-thickness surgical ablation, using biopsy punches, and to remove a large volume of the tibialis anterior muscle of the lower limb in mice. This procedure results in a reduction in muscle mass and limited regeneration capacity; the approach is easy to reproduce and can also be applied to larger animal models. For therapeutic applications, we further explain how to implant bioscaffolds into the ablated muscle site. With adequate training and practice, the surgical procedure can be performed within 30 min.

Medical devices are a mainstay of the healthcare industry, providing clinicians with innovative tools to diagnose, monitor, and treat a range of medical conditions. For implantable devices, it is widely regarded that chronic inflammation during the foreign body response (FBR) is detrimental to device performance, but also required for tissue regeneration and host integration. Current strategies to mitigate the FBR rely on broad acting anti‐inflammatory drugs, most commonly, dexamethasone (DEX), which can inhibit angiogenesis and compromise long‐term device function. This study challenges prevailing assumptions by suggesting that FBR inflammation is multifaceted, and selectively targeting its individual pathways can stop implant fibrosis while preserving beneficial repair pathways linked to improved device performance. MCC950, an anti‐inflammatory drug that selectively inhibits the NLRP3 inflammasome, targets pathological inflammation without compromising global immune function. The effects of MCC950 and DEX on the FBR are compared using implanted polycaprolactone (PCL) scaffolds. The results demonstrate that both DEX and MCC950 halt immune cell recruitment and cytokine release, leading to reduced FBR. However, MCC950 achieves this while supporting capillary growth and enhancing tissue angiogenesis. These findings support selective immunosuppression approaches as a potential future direction for treating the FBR and enhancing the longevity and safety of implantable devices.

Murine myoblasts cultured on combinatorial extracellular matrix (ECM) proteins are exposed to uniaxial strain. The combined effects of ECMs and strain on myogenesis are investigated by transcriptomic and protein analyses.

Volumetric muscle loss (VML) is associated with irreversibly impaired muscle function due to traumatic injury. Experimental approaches to treat VML include the delivery of basic fibroblast growth factor (bFGF) or rehabilitative exercise. The objective of this study was to compare the effects of spatially nanopatterned collagen scaffold implants with either bFGF delivery or in conjunction with voluntary exercise. Aligned nanofibrillar collagen scaffold bundles were adsorbed with bFGF, and the bioactivity of bFGF-laden scaffolds was examined by skeletal myoblast or endothelial cell proliferation. The therapeutic efficacy of scaffold implants with either bFGF release or exercise was examined in a murine VML model. Our results show an initial burst release of bFGF from the scaffolds, followed by a slower release over 21 days. The released bFGF induced myoblast and endothelial cell proliferation in vitro. After 3 weeks of implantation in a mouse VML model, twitch force generation was significantly higher in mice treated with bFGF-laden scaffolds compared to bFGF-laden scaffolds with exercise. However, myofiber density was not significantly improved with bFGF scaffolds or voluntary exercise. In contrast, the scaffold implant with exercise induced more re-innervation than all other groups. These results highlight the differential effects of bFGF and exercise on muscle regeneration.

Multifunctional nanocarriers (MNCs) promise to improve therapeutic outcomes by combining multiple classes of molecules into a single nanostructure, enhancing active targeting of therapeutic agents and facilitating new combination therapies. However, nanocarrier platforms currently approved for clinical use can still only carry a single therapeutic agent. The complexity and escalating costs associated with the synthesis of more complex MNCs have been major technological roadblocks in the pathway for clinical translation. Here, we show that plasma polymerized nanoparticles (PPNs), synthesised in reactive gas discharges, can bind and effectively deliver multiple therapeutic cargo in a facile and cost-effective process compatible with up scaled commercial production. Delivery of siRNA against vascular endothelial growth factor (siVEGF) at extremely low concentrations (0.04 nM), significantly reduced VEGF expression in hard-to-transfect cells when compared with commercial platforms carrying higher siRNA doses (6.25 nM). PPNs carrying a combination of siVEGF and standard of care Paclitaxel (PPN-Dual) at reduced doses (< 100 µg/kg) synergistically modulated the microenvironment of orthotopic breast tumors in mice, and significantly reduced tumor growth. We propose PPNs as a new nanomaterial for delivery of therapeutics, which can be easily functionalised in any laboratory setting without the need for additional wet-chemistry and purification steps.

Although stem cell therapy has tremendous therapeutic potential, clinical translation of stem cell therapy has yet to be fully realized. Recently, patient comorbidities and lifestyle choices have e...

The functionality and durability of implanted biomaterials are often compromised by an exaggerated foreign body reaction (FBR). M1/M2 polarization of macrophages is a critical regulator of scaffold induced FBR. Macrophage colony-stimulating factor (M-CSF), a haematopoietic growth factor, induces macrophages into an M2-like polarized state, leading to immunoregulation and promoting tissue repair. In the present study, we explored the immunomodulatory effects of surface bound M CSF on Poly-L-lactic acid (PLLA)-induced FBR. M-CSF was immobilised on the surface of PLLA via plasma immersion ion implantation (PIII). M-CSF functionalized PLLA, PLLA-only, and PLLA+PIII were assessed in an IL-1β luciferase reporter mouse to detect real-time levels of IL 1β expression, reflecting acute inflammation in vivo. Additionally, these different treated scaffolds were implanted subcutaneously into wild-type mice to explore the effect of M-CSF in polarization of M2-like macrophages (CD68+/CD206+), related cytokines (pro-inflammatory: IL-1β, TNF and MCP-1; anti-inflammatory: IL-10 and TGF-β) and angiogenesis (CD31) by immunofluorescent staining. Our data demonstrated that IL-1β activity in M-CSF functionalized scaffolds was ~ 50% reduced compared to PLLA-only at day 1 (p < 0.01) and day 2 (p < 0.05) post-implantation. CD206+ macrophages were >2.6-fold more in M-CSF functionalized PLLA compared to PLLA-only at day 7 (p < 0.001), along with higher levels of IL-10 at both day 7 (p < 0.05) and day 14 (p < 0.01), and TGF-β at day 3 (p < 0.05), day 7 (p < 0.05) and day 14 (p < 0.001). Lower levels of pro-inflammatory cytokines were also detected in M CSF functionalized PLLA in the early-phase of the immune response compared to PLLA-only: a ~58% decrease at day 3 in IL-1β; a ~91% decrease at day 3 and a ~66% decrease at day 7 in TNF; a ~ 60% decrease at day 7 in MCP-1. Moreover, enhanced angiogenesis inside and on/near the scaffold was observed in M-CSF functionalized PLLA compared to PLLA-only at day 3 (p < 0.05) and day7 (p < 0.05), respectively. Overall, M CSF functionalized PLLA enhanced CD206+ macrophages polarization and angiogenesis, consistent with lower levels of pro-inflammatory cytokines and higher levels of anti-inflammatory cytokines in early stages of the host response, indicating potential immunoregulatory functions on the local environment.