Investigating macrophage plasticity emerges as a promising strategy for promoting tissue regeneration and can be exploited by regulating the transient receptor potential vanilloid 4 (TRPV4) channel. The TRPV4 channel responds to various stimuli including mechanical, chemical, and selective pharmacological compounds. It is well documented that treating cells such as epithelial cells and fibroblasts with a TRPV4 agonist enhances the Ca²⁺ influx to the cells, which leads to secretion of pro-inflammatory cytokines, while a TRPV4 antagonist reduces both Ca²⁺ influx and pro-inflammatory cytokine secretion. In this work, we investigated the effect of selective TRPV4 modulator compounds on U937-differentiated macrophages encapsulated within three-dimensional (3D) matrices. Despite offering a more physiologically relevant model than 2D cultures, pharmacological treatment of macrophages within 3D collagen matrices is largely overlooked in the literature. In this study, pro-inflammatory macrophages were treated with an agonist, 500 nM of GSK1016790A (TRPV4(+)), and an antagonist, 10 mM of RN-1734 (TRPV4(−)), to elucidate the modulation of the TRPV4 channel at both cellular and extracellular levels. To evaluate macrophage phenotypic alterations within 3D collagen matrices following TRPV4 modulator treatment, we employed structural techniques (SEM, Masson’s trichrome, and collagen hybridizing peptide (CHP) staining), quantitative morphological measures for phenotypic assessment, and genotypic methods such as quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). Our data reveal that pharmacological modulation of the macrophage TRPV4 channel alters the cytoskeletal structure of macrophages and influences the 3D structure encapsulating them. Moreover, we proved that treating macrophages with a TRPV4 agonist and antagonist enhances the expression of pro- and anti-inflammatory genes, respectively, leading to the upregulation of surface markers CD80 and CD206. In the TRPV4(−) group, the CD206 gene and CD206 surface marker were significantly upregulated by 9- and 2.5-fold, respectively, compared to the control group. These findings demonstrate that TRPV4 modulation can be utilized to shift macrophage phenotype within the 3D matrix toward a desired state. This is an innovative approach to addressing inflammation in musculoskeletal tissues.

Lower back pain commonly arises from intervertebral disc (IVD) failure, often caused by deteriorating annulus fibrosus (AF) and/or nucleus pulposus (NP) tissue. High socioeconomic cost, quality of life issues, and unsatisfactory surgical options motivate the rapid development of non-invasive, regenerative repair strategies for lower back pain. This study aims to evaluate the AF regenerative capacity of injectable matrix repair strategy in ex vivo porcine organ culturing using collagen type-I and polycaprolactone nanofibers (PNCOL) with encapsulated fibroblast cells. Upon 14 days organ culturing, the porcine IVDs were assessed using gross optical imaging, magnetic resonance imaging (MRI), histological analysis, and Reverse Transcriptase quantitative PCR (RT-qPCR) to determine the regenerative capabilities of the PNCOL matrix at the AF injury. PNCOL-treated AF defects demonstrated a full recovery with increased gene expressions of AF extracellular matrix markers, including Collagen-I, Aggrecan, Scleraxis, and Tenascin, along with anti-inflammatory markers such as CD206 and IL10. The PNCOL treatment effectively regenerates the AF tissue at the injury site contributing to decreased herniation risk and improved surgical outcomes, thus providing effective non-invasive strategies for treating IVD injuries.

Mechano-rehabilitation, also known as mechanotherapy, represents the forefront of noninvasive treatment for musculoskeletal tissue (MSK) disorders, encompassing conditions affecting tendons, cartilage, ligaments, and muscles. Recent emphasis has underscored the significance of macrophage presence in the healing of MSK tissues. However, a considerable gap still exists in comprehending how mechanical strains associated with mechanotherapy impact both the naïve and pro-inflammatory macrophage phenotypes within the 3D tissue matrix, as well as whether the shift in macrophage phenotype is contingent upon the mechanical strains inherent to mechanotherapy. In this study, we delineated alterations in mechanoadaptation and polarization of both naïve and M1 macrophages within 3D matrices, elucidating their response to varying degrees of mechanical strain exposure (3%, 6%, and 12%). To evaluate macrophage mechanoadaptation and mechanosensitivity within 3D collagen matrices under mechanical loading, we employed structural techniques (SEM, histology), quantitative morphological measures for phenotypic assessment, and genotypic methods such as quantitative real-time PCR (qRT-PCR). Our data reveal that the response of macrophages to mechanical loading is not only contingent upon their specific sub-phenotype but also varies with the amplitude of mechanical strain. Notably, while supra-mechanical loading (12% strain) was requisite to induce a phenotypic shift in naïve (M0) macrophages, as little as 3% mechanical strain proved sufficient to prompt phenotypic alterations in pro-inflammatory (M1) macrophages. These findings pave the way for leveraging the macrophage mechanome in customized and targeted applications of mechanical strain within the mechanotherapeutic framework. Considering the prevalence of MSK tissue injuries and their profound societal and economic implications, the development of well-informed and effective clinical mechanotherapy modalities for MSK tissue healing becomes an imperative endeavor.

In periodontitis, the bone remodeling process is disrupted by the prevalent involvement of bacteria-induced pro-inflammatory macrophage cells and their interaction between osteoblast cells residing within the infected bone tissue. The complex interaction between the cells needs to be deciphered to understand the dominant player in tipping the balance from osteogenesis to osteoclastogenesis. Yet, only a few studies have looked at the crosstalk interaction between osteoblasts and macrophages using biomimetic three-dimensional (3D) tissue-like matrices. In this study, we created a cell-laden 3D tissue analog to study indirect crosstalk between these two cell types and the direct synergistic effect when they were cultured together on a 3D scaffold. The cell-specific role on osteoclast differentiation was investigated through osteoblast- and pro-inflammatory macrophage-specific feedback studies. The results suggested that when macrophages were exposed to osteoblasts-derived conditioned media from the mineralized matrix, the M1 macrophages tended to maintain their pro-inflammatory phenotype. Further, when osteoblasts were exposed to secretions from pro-inflammatory macrophages, they demonstrated elevated Receptor activator of NF-kB ligand (RANKL) expression and decreased ALP activities compared to osteoblasts exposed to only osteogenic media. In addition, the upregulation of TNF-α and c-Fos in pro-inflammatory macrophages within the 3D matrix indirectly increased the RANKL expression and reduced the ALP activity of osteoblasts, which in turn promoted osteoclastogenesis. The contact co-culturing with osteoblast and pro-inflammatory macrophages within 3D matrix demonstrated that the pro-inflammatory markers (TNFα and IL1β) expressions were upregulated. In contrast, anti-inflammatory marker (CCL18) were downregulated and osteoclastic markers (TRAP 6 and ACP5) were unchanged. The data suggested that the osteoblasts curbed the osteoclastic differentiation of macrophages while macrophages still preserve their pro-inflammatory lineages. The osteoblast within the 3D co-culture demonstrated increased ALP activity and did not express RANKL significantly different than the osteoblast cultured within a 3D collagen matrix without macrophages. Contact co-culturing has an anabolic effect on bone tissue in a bacteria-derived inflammatory environment.

This study aimed to understand extracellular mechanical stimuli’s effect on prostate cancer cells’ metastatic progression within a three-dimensional (3D) bone-like microenvironment. In this study, a mechanical loading platform, EQUicycler, has been employed to create physiologically relevant static and cyclic mechanical stimuli to a prostate cancer cell (PC-3)-embedded 3D tissue matrix. Three mechanical stimuli conditions were applied: control (no loading), cyclic (1% strain at 1 Hz), and static mechanical stimuli (1% strain). The changes in prostate cancer cells’ cytoskeletal reorganization, polarity (elongation index), proliferation, expression level of N-Cadherin (metastasis-associated gene), and migratory potential within the 3D collagen structures were assessed upon mechanical stimuli. The results have shown that static mechanical stimuli increased the metastasis progression factors, including cell elongation (p < 0.001), cellular F-actin accumulation (p < 0.001), actin polymerization (p < 0.001), N-Cadherin gene expression, and invasion capacity of PC-3 cells within a bone-like microenvironment compared to its cyclic and control loading counterparts. This study established a novel system for studying metastatic cancer cells within bone and enables the creation of biomimetic in vitro models for cancer research and mechanobiology.

Extracellular vesicles (EVs) have emerged as promising acellular tools for modulating immune responses for tissue engineering applications. This study explores the potential of human fibroblast-derived EVs delivered within a three-dimensional (3D) injectable scaffold composed of polycaprolactone (PCL) nanofibers and collagen (PNCOL) to reprogram macrophage behavior and support scaffold integrity under inflammatory conditions. EVs were successfully isolated from human fibroblasts using ultracentrifugation and characterized for purity, size distribution and surface markers (CD63 and CD9). Macrophage-laden PNCOL scaffolds were prepared under three conditions: macrophage-only (MP), fibroblast co-encapsulated (F-MP), and EV-encapsulated (EV-MP) groups. Structural integrity was assessed via scanning electron microscopy and Masson’s trichrome staining, while immunomodulatory effects were evaluated through metabolic assays, gene expression profiling, and immunohistochemistry for macrophage polarization markers (CD80, CD206). When co-encapsulated with pro-inflammatory (M1) macrophages in PNCOL scaffolds, fibroblast-derived EVs preserved scaffold structure and significantly enhanced macrophage metabolic activity compared to the control (MP) and other experimental group (F-MP). The gene expression and immunohistochemistry data demonstrated substantial upregulation of anti-inflammatory markers (TGF-β, CD163, and CCL18) and surface protein CD206, indicating a phenotypic shift toward M2-like macrophages for EV-encapsulated scaffolds relative to the other groups. The findings of this study demonstrate that fibroblast-derived EVs integrated into injectable PCL–collagen scaffolds offer a viable, cell-free approach to modulate inflammation, preserve scaffold structure, and support regenerative healing. This strategy holds significant promise for advancing immuno-instructive platforms in regenerative medicine, particularly in settings where conventional cell therapies face limitations in survival, cost, or safety.